Penicillium purpurogenum mutanases and nucleic acids encoding same

ABSTRACT

The present invention relates to polypeptides having mutanase activity and isolated nucleic acid sequences encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the nucleic acid sequences as well as methods for producing the polypeptides. The present invention further relates to oral cavity compositions and methods for degrading mutan.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of U.S. application Ser. No.08/598,881 filed Feb. 9, 1996, now abandoned which application is fullyincorporated herein by reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to polypeptides having mutanase activityand isolated nucleic acid sequences encoding the polypeptides. Theinvention also relates to nucleic acid constructs, vectors, and hostcells comprising the nucleic acid sequences as well as methods forproducing the polypeptides. The invention further relates tocompositions comprising the polypeptides and methods of use thereof.

2. Description of the Related Art

The formation of dental plaque leads to dental caries, gingivalinflammation, periodontal disease, and eventually tooth loss. Dentalplaque is a mixture of bacteria, epithelial cells, leukocytes,macrophages, and other oral exudate. The bacteria produce glucans andlevans from sucrose found in the oral cavity. These glucans, levans, andmicroorganisms form an adhesive matrix for the continued proliferationof plaque.

Streptococcus mutans is a common bacterium associated with dentalplaque. Extracellular insoluble polysaccharides produced by thisbacterium in the oral cavity play an important role for adhesion andproliferation of bacteria on the surface of teeth and, hence, may beimportant in the etiology of dental caries. Mutan is the major componentof the insoluble polysaccharides produced by Streptococcus mutans and iscomprised of a backbone with α-1,3-glycosidic linkages and branches withα-1,6-glycosidic linkages.

Mutanases are α-1,3-glucanases (also known as α-1,3-glucanohydrolases)which degrade the α-1,3-glycosidic linkages in mutan. Mutanases havebeen described from two species of Trichoderma (Hasegawa et al., 1969,Journal of Biological Chemistry 244:5460-5470; Guggenheim and Haller,1972, Journal of Dental Research 51:394-402) and from a strain ofStreptomyces (Takehara et al., 1981, Journal of Bacteriology145:729-735). A mutanase gene from Trichodenna harzianum has been clonedand sequenced (Japanese Patent No. 4-58889/A).

Although mutanases have commercial potential for use as an antiplaqueagent in dental applications and personal care products, e.g.,toothpaste, chewing gum, or other oral and dental care products, the arthas been unable to produce mutanases in significant quantities to becommercially useful.

It is an object of the present invention to provide new mutanases whichcan be produced in commercially useful quantities.

SUMMARY OF THE INVENTION

The present invention relates to isolated polypeptides having mutanaseactivity selected from the group consisting of:

(a) a polypeptide with an amino acid sequence set forth in SEQ ID NO:3;

(b) a polypeptide which is encoded by a nucleic acid sequence which iscapable of hybridizing under high stringency conditions with (i) thenucleic acid sequence set forth in SEQ ID NO:2, or (ii) itscomplementary strand;

(c) a polypeptide with an amino acid sequence which has at least 60%identity with the amino acid sequence set forth in SEQ ID NO:3;

(d) an allelic form of (a), (b), or (c); and

(e) a fragment of (a), (b), (c), or (d).

The present invention also relates to isolated nucleic acid sequencesencoding the polypeptides and to nucleic acid constructs, vectors, andhost cells comprising the nucleic acid sequences as well as methods forproducing the polypeptides. The present invention further relates tooral cavity compositions and methods for degrading mutan.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the hybridization analysis of Penicillium purpurogenumgenomic DNA with a Trichoderma harzianum cDNA probe.

FIG. 2 shows a partial restriction map of a 3.6 kb DNA insert in clonePp6A.

FIG. 3 shows the genomic DNA sequence and deduced amino acid sequence ofPenicillium purpurogenum CBS 238.95 mutanase (SEQ ID NO:2 and SEQ IDNO:3, respectively).

FIG. 4 shows the alignment of the amino acid sequences for thePenicillium purpurogenum CBS 238.95 mutanase and the Trichodermaharzianum mutanase (SEQ ID NO:5).

FIG. 5 shows a restriction map of pBANe6.

FIG. 6 shows the pH profile of the Penicillium purpurogenum CBS 238.95mutanase.

FIG. 7 shows the temperature profile of the Penicillium purpurogenum CBS238.95 mutanase.

DETAILED DESCRIPTION OF THE INVENTION

Polypeptides Having Mutanase Activity

In a first embodiment, the present invention relates to isolatedpolypeptides having mutanase activity with the amino acid sequence setforth in SEQ ID NO:3 or a fragment or subsequence thereof which retainsmutanase activity. Preferably, a fragment contains at least 400 aminoacid residues, more preferably at least 475 amino acid residues, evenmore preferably at least 550 amino acid residues, and most preferably atleast 600 amino acid residues.

The polypeptides of the present invention are preferably obtained fromspecies of Penicillium including, but not limited to, Penicilliumallahabadense, Penicillium arenicola, Penicillium asperum, Penicilliumaurantiogriseum, Penicillium bilaii, Penicillium brevicompactum,Penicillium camembertii, Penicillium canescens, Penicillium chrysogenum,Penicillium citreonigrum, Penicillium citreoviride, Penicilliumcitrinum, Penicillium claviforme, Penicillium commune, Penicilliumconcentricum, Penicillium corylophilum, Penicillium corymbiferum,Penicillium crustosum, Penicillium cyclopium, Penicillium decumbens,Penicillium digitatum, Penicillium diversum, Penicillium duclauxii,Penicillium echinulatum, Penicillium expansum, Penicillium fellutanum,Penicillium frequentans, Penicillium funiculosum, Penicillium glabrum,Penicillium glandicola, Penicillium granulatum, Penicilliumgriseofulvum, Penicillium hirsutum, Penicillium hordei, Penicilliumimplicatum, Penicillium islandicum, Penicillium italicum, Penicilliumjanczewskii, Penicillium janthinellum, Penicillium lividum, Penicilliumluteum, Penicillium melinii, Penicillium miczynskii, Penicilliumminioluteum, Penicillium montanense, Penicillium nigricans, Penicilliumolivicolor, Penicillium olsonii, Penicillium oxalicum, Penicilliumpiceum, Penicillium pinophilum, Penicillium puberulum, Penicilliumpurpurogenum (synonymous with Penicillium rubrum), Penicillium pusillum,Penicillium raciborskii, Penicillium raistrickii, Penicilliumrestrictum, Penicillium roqueforti, Penicillium rugulosum, Penicilliumsclerotiorum, Penicillium simplicissimum, Penicillium spiculisporum,Penicillium spinulosum, Penicillium stipitatum, Penicillium striatum,Penicillium terlikowskii, Penicillium thomii, Penicillium variabile,Penicillium varians, Penicillium vermiculatum, Penicillium verrucosum,Penicillium viridicatum, Penicillium vulpinum, Penicillium urticae,Penicillium waksmanii, and Penicillium wortmanni. Strains of thesespecies are readily accessible to the public in a number of culturecollections, such as the American Type Culture Collection (ATCC),Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSM),Centraalbureau Voor Schimmelcultures (CBS), and Agricultural ResearchService Patent Culture Collection, Northern Regional Research Center(NRRL).

In a more preferred embodiment, a polypeptide of the present inventionis obtained from Penicillium purpurogenum , and most preferably fromPenicillium purpurogenum CBS 238.95 or a mutant strain thereof, e.g.,the polypeptide with the amino acid sequence set forth in SEQ ID NO:3.

A polypeptide of the present invention may also be obtained fromteleomorphs of Penicillium, e.g., Eupenicillium and Talaromyces,including, but not limited to, Eupenicillium alutaceum, Eupenicilliumcinnamopurpureum, Eupenicillium crustaceum, Eupenicillium hirayamae,Eupenicillium pinetorum, Eupenicillium javanicum, Eupenicilliumlapidosum, Eupenicillium ludwigii, Eupenicillium ochrosalmoneum,Eupenicillium shearii, Talaromyces flavus, Talaromyces stipitatus,Talaromyces luteus, Talaromyces wortmanii, Talaromyces trachyspernus,Talaromyces thennophilus,and Talaromyces striatus.

A polypeptide of the present invention may further be obtained fromother fungi which are synonyms of Penicillium as defined by Samson andPitt In Samson and Pitt (eds.), Advances in Penicillium and AspergillusSystematics, Plenum Press, ASI Series, New York, 1985. Penicillium is agenus of Hyphomycetes, characterized by the production of conidia, whichare usually green, in chains from verticils of phialides. Phialides maybe directly supported on a stipe or on one, two, or rarely three compactstages of supporting cells: metulae and rami in that order, with ramuliin between on occasion. Phialides have short straight necks and smoothwalls, and are characteristically produced on a stipe or a metula over aperiod of time, not simultaneously.

For purposes of the present invention, the term "obtained from" as usedherein in connection with a given source shall mean that the polypeptideis produced by the source or by a cell in which a gene from the sourcehas been inserted.

In a second embodiment, the present invention relates to polypeptideswhich are encoded by nucleic acid sequences which are capable ofhybridizing under high stringency conditions with an oligonucleotideprobe which hybridizes under the same conditions with the nucleic acidsequence set forth in SEQ ID NO:2 or its complementary strand (J.

Sambrook, E. F. Fritsch, and T. Maniatus, 1989, Molecular Cloning, ALaboratory Manual, 2d edition, Cold Spring Harbor, N.Y.). Hybridizationindicates that the analogous is nucleic acid sequence hybridizes to theoligonucleotide probe corresponding to the polypeptide encoding part ofthe nucleic acid sequence shown in SEQ ID NO:2, under low to highstringency conditions (for example, prehybridization and hybridizationat 42° C. in 5X SSPE, 0.3% SDS, 200 μg/ml sheared and denatured salmonsperm DNA, and either 50, 35 or 25% formamide for high, medium and lowstringencies, respectively), following standard Southern blottingprocedures.

SEQ ID NO:2 may be used to identify and clone DNA encoding polypeptideshaving mutanase activity from other strains of different genera orspecies according to methods well known in the art. Thus, a genomic,cDNA or combinatorial chemical library prepared from such otherorganisms may be screened for DNA which hybridizes with SEQ ID NO:2 andencodes mutanase. Genomic or other DNA from such other organisms may beseparated by agarose or polyacrylamide gel electrophoresis, or otherseparation techniques. DNA from the libraries or the separated DNA maybe transferred to and immobilized on nitrocellulose or other suitablecarrier material. In order to identify clones or DNA which is homologouswith SEQ ID NO:2, the carrier material is used in a Southern blot inwhich the carrier material is finally washed three times for 30 minuteseach using 2XSSC, 0.2% SDS at preferably not higher than 50° C., morepreferably not higher than 55° C., more preferably not higher than 60°C., and even more preferably not higher than 65° C. Molecules to whichthe oligonucleotide probe hybridizes under these conditions are detectedusing X-ray film.

In a third embodiment, the present invention relates to polypeptideswhich have an amino acid sequence which has a degree of identity to theamino acid sequence set forth in SEQ ID NO:3 of at least about 60%,preferably at least about 70%, more preferably at least about 80%, evenmore preferably at least about 90%, most preferably at least 95%, andeven most preferably at least about 97%, which qualitatively retain themutanase activity of the polypeptides (hereinafter "homologouspolypeptides"). In a preferred embodiment, the homologous polypeptideshave an amino acid sequence which differs by five amino acids,preferably by four amino acids, more preferably by three amino acids,even more preferably by two amino acids, and most preferably by oneamino acid from the amino acid sequence set forth in SEQ ID NO:3. Thedegree of identity between two or more amino acid sequences may bedetermined by means of computer programs known in the art such as GAPprovided in the GCG program package (Needleman and Wunsch, 1970, Journalof Molecular Biology 48:443-453). For purposes of determining the degreeof identity between two amino acid sequences for the present invention,the Clustal method (Higgins, 1989, CABIOS 5:151-153) is used with anidentity table, a gap penalty of 10, and a gap length of 10.

The amino acid sequences of the homologous polypeptides differ from theamino acid sequence set forth in SEQ ID NO:3 by an insertion or deletionof one or more amino acid residues and/or the substitution of one ormore amino acid residues by different amino acid residues. Preferably,amino acid changes are of a minor nature, that is conservative aminoacid substitutions that do not significantly affect the folding and/oractivity of the protein; small deletions, typically of one to about 30amino acids; small amino- or carboxyl-terminal extensions, such as anamino-terminal methionine residue; a small linker peptide of up to about20-25 residues; or a small extension that facilitates purification bychanging net charge or another function, such as a poly-histidine tract,an antigenic epitope or a binding domain.

Examples of conservative substitutions are within the group of basicamino acids (such as arginine, lysine and histidine), acidic amino acids(such as glutamic acid and aspartic acid), polar amino acids (such asglutamine and asparagine), hydrophobic amino acids (such as leucine,isoleucine and valine), aromatic amino acids (such as phenylalanine,tryptophan and tyrosine), and small amino acids (such as glycine,alanine, serine, threonine and methionine). Amino acid substitutionswhich do not generally alter the specific activity are known in the artand are described, e.g., by H. Neurath and R. L. Hill, 1979, In, TheProteins, Academic Press, New York. The most commonly occurringexchanges are: Ala/Ser, Val/Ile, Asp/Glu, Thr/Ser, Ala/Gly, Ala/Thr,Ser/Asn, Ala/Val, Ser/Gly, Tyr/Phe, Ala/Pro, Lys/Arg, Asp/Asn, Leu/Ile,Leu/Val, Ala/Glu, Asp/Gly as well as these in reverse.

The present invention also relates to polypeptides having immunochemicalidentity or partial immunochemical identity to the polypeptide native toPenicillium purpurogenum CBS 238.95. In this embodiment, a polypeptideof the present invention is used to produce antibodies which areimmunoreactive or bind to epitopes of the polypeptide. A polypeptidehaving immunochemical identity to the polypeptide native to Penicilliumpurpurogenum CBS 238.95 means that an antiserum containing antibodiesagainst the polypeptide native to Penicillium purpurogenum CBS 238.95reacts with the other polypeptide in an identical fashion such as totalfusion of precipitates, identical precipitate morphology, and/oridentical electrophoretic mobility using a specific immunochemicaltechnique. A further explanation of immunochemical identity is describedby Axelsen, Bock, and Kr.o slashed.ll, In N. H. Axelsen, J. Kr.oslashed.ll, and B. Weeks, editors, A Manual of QuantitativeImmunoelectrophoresis, Blackwell Scientific Publications, 1973, Chapter10. Partial immunochemical identity means that an antiserum containingantibodies against the polypeptide native to Penicillium purpurogenumCBS 238.95 reacts with the other polypeptide in a partially identicalfashion such as partial fusion of precipitates, partially identicalprecipitate morphology, and/or partially identical electrophoreticmobility using a specific immunochemical technique. A furtherexplanation of partial immunochemical identity is described by Bock andAxelsen, In N. H. Axelsen, J. Kr.o slashed.ll, and B. Weeks, editors, AManual of Quantitative Immunoelectrophoresis, Blackwell ScientificPublications, 1973, Chapter 11. The immunochemical properties aredetermined by immunological cross-reaction identity tests by thewell-known Ouchterlony double immunodiffusion procedure. Specifically,an antiserum against the polypeptide of the invention is raised byimmunizing rabbits (or other rodents) according to the proceduredescribed by Harboe and Ingild, In N. H. Axelsen, J. Kr.o slashed.ll,and B. Weeks, editors, A Manual of Quantitative Immunoelectrophoresis,Blackwell Scientific Publications, 1973, Chapter 23, or Johnstone andThorpe, Immunochemistry in Practice, Blackwell Scientific Publications,1982 (more specifically pages 27-31).

Polypeptides which are encoded by nucleic acid sequences which arecapable of hybridizing with an oligonucleotide probe which hybridizeswith the nucleic acid sequence set forth in SEQ ID NO:2, itscomplementary strand or a subsequence thereof, the homologouspolypeptides and polypeptides having identical or partially identicalimmunological properties may be obtained from microorganisms of anygenus, preferably from a bacterial or fungal source. Sources for suchpolypeptides are strains of the genus Penicillium and species thereofavailable in public depositories. Furthermore, such polypeptides may beidentified and obtained from other sources including microorganismsisolated from nature (e.g., soil, composts, water, etc.) using theabove-mentioned probes. Techniques for isolating microorganisms fromnatural habitats are well known in the art. The nucleic acid sequencemay then be derived by similarly screening a cDNA library of anothermicroorganism, in particular a fungus, such as a strain of anAspergillus sp., in particular a strain of Aspergillus aculeatus,Aspergillus awamori, Aspergillus foetidus, Aspergillus japonicus,Aspergillus nidulans, Aspergillus niger or Aspergillus oryzae, a strainof Trichoderma sp., in particular a strain of Trichoderma harzianum ,Trichodenna koningii, Trichodenna longibrachiatum, Trichodenna reesei orTrichodenna viride, or a strain of a Fusarium sp., in particular astrain of Fusarium cerealis, Fusarium crookwellense, Fusariumgraminearum, Fusarium oxysporum, Fusarium sambucinum or Fusariumsulphureum, or a strain of a Humicola sp., or a strain of anAureobasidium sp., a Cryptococcus sp., a Filibasidium sp., a Magnaporthesp., a Myceliophthora sp., a Neocallimastix sp., a Paecilomyces sp., aPiromyces sp., a Talaromyces sp., a Thermoascus sp., a Thielavia sp., ora Schizophyllum sp. Once a nucleic acid sequence encoding a polypeptidehas been detected with the probe(s), the sequence may be isolated orcloned by utilizing techniques which are known to those of ordinaryskill in the art (see, e.g., Sambrook et al., supra).

As defined herein, an "isolated" polypeptide is a polypeptide which isessentially free of other non-mutanase polypeptides, e.g., at leastabout 20% pure, preferably at least about 40% pure, more preferablyabout 60% pure, even more preferably about 80% pure, most preferablyabout 90% pure, and even most preferably about 95% pure, as determinedby SDS-PAGE.

The present invention also relates to hybrid or fusion polypeptides,comprising the catalytic domain included in the amino acid sequence setforth in SEQ ID NO:3. In a preferred embodiment, these polypeptides havemutanase activity.

The present invention also relates to hybrid or fusion polypeptides,comprising the linker included in the amino acid sequence set forth inSEQ ID NO:3. In a preferred embodiment, these polypeptides have mutanaseactivity.

The present invention also relates to hybrid or fusion polypeptides,comprising the mutan binding domain included in the amino acid sequenceset forth in SEQ ID NO:3. In a preferred embodiment, these polypeptideshave mutanase activity.

Nucleic Acid Sequences

The present invention also relates to isolated nucleic acid sequenceswhich encode a polypeptide of the present invention. In a preferredembodiment, the nucleic acid sequence encodes a polypeptide obtainedfrom Penicillium, e.g., Penicillium purpurogenum , and in a morepreferred embodiment, the nucleic acid sequence is obtained fromPenicillium purpurogenum CBS 238.95, e.g., the nucleic acid sequence setforth in SEQ ID NO:2. In a more preferred embodiment, the nucleic acidsequence is the sequence contained in plasmid pZL-Pp6A which iscontained in Escherichia coli NRRL B-21518. The present invention alsoencompasses nucleic acid sequences which encode a polypeptide having theamino acid sequence set forth in SEQ ID NO:3, which differ from SEQ IDNO:2 by virtue of the degeneracy of the genetic code. The presentinvention also relates to subsequences of SEQ ID NO:2 which encode afragment of SEQ ID NO:3 which retains mutanase activity. Preferably, asubsequence of SEQ ID NO:2 which encodes a fragment of SEQ ID NO:3 whichretains mutanase activity contains at least 1400 nucleotides, morepreferably at least 1650 nucleotides, and most preferably at least 1800nucleotides.

As described above, the nucleic acid sequences may be obtained frommicroorganisms which are synonyms or teleomorphs of Penicillium asdefined by Samson and Pitt, 1985, supra.

The techniques used to isolate or clone a nucleic acid sequence encodinga polypeptide are known in the art and include isolation from genomicDNA, preparation from cDNA, or a combination thereof. The cloning of thenucleic acid sequences of the present invention from such genomic DNAcan be effected, e.g., by using the well known polymerase chain reaction(PCR) or antibody screening of expression libraries to detect cloned DNAfragments with shared structural features. See, e.g., Innis et al.,1990, A Guide to Methods and Application, Academic Press, New York.Other nucleic acid amplification procedures such as ligase chainreaction (LCR), ligated activated transcription (LAT) and nucleic acidsequence-based amplification (NASBA) may be used. The nucleic acidsequence may be cloned from a strain of the Penicillium producing thepolypeptide, or another or related organism and thus, for example, maybe an allelic or species variant of the polypeptide encoding region ofthe nucleic acid sequence.

The term "isolated" nucleic acid sequence as used herein refers to anucleic acid sequence which is essentially free of other nucleic acidsequences, e.g., at least about 20% pure, preferably at least about 40%pure, more preferably about 60% pure, even more preferably about 80%pure, most preferably about 90% pure, and even most preferably about 95%pure, as determined by agarose gel electrophoresis. For example, anisolated nucleic acid sequence can be obtained by standard cloningprocedures used in genetic engineering to relocate the nucleic acidsequence from its natural location to a different site where it will bereproduced. The cloning procedures may involve excision and isolation ofa desired nucleic acid fragment comprising the nucleic acid sequenceencoding the polypeptide, insertion of the fragment into a vectormolecule, and incorporation of the recombinant vector into a host cellwhere multiple copies or clones of the nucleic acid sequence will bereplicated. The nucleic acid sequence may be of genomic, cDNA, RNA,semisynthetic, synthetic origin, or any combinations thereof.

The present invention also relates to nucleic acid sequences which havea nucleic acid sequence which has a degree of identity to the nucleicacid sequence set forth in SEQ ID NO:2 of at least about 60%, preferablyat least about 70%, more preferably at least about 80%, even morepreferably at leat about 90%, most preferably at least about 95 %, andeven most preferably at least about 97%, which encode an activepolypeptide. The degree of identity between two nucleic acid sequencesmay be determined by means of computer programs known in the art such asGAP provided in the GCG program package (Needleman and Wunsch, 1970,Journal of Molecular Biology 48:443-453). For purposes of determiningthe degree of identity between two nucleic acid sequences for thepresent invention, the Clustal method (Higgins, 1989, supra) is usedwith an identity table, a gap penalty of 10, and a gap length of 10.

Modification of the nucleic acid sequence encoding the polypeptide maybe necessary for the synthesis of polypeptides substantially similar tothe polypeptide. The term "substantially similar" to the polypeptiderefers to non-naturally occurring forms of the polypeptide. Thesepolypeptides may differ in some engineered way from the polypeptideisolated from its native source. For example, it may be of interest tosynthesize variants of the polypeptide where the variants differ inspecific activity, thermostability, pH optimum, or the like using, e.g.,site-directed mutagenesis. The analogous sequence may be constructed onthe basis of the nucleic acid sequence presented as the polypeptideencoding part of SEQ ID NO:2, e.g., a sub-sequence thereof, and/or byintroduction of nucleotide substitutions which do not give rise toanother amino acid sequence of the polypeptide encoded by the nucleicacid sequence, but which corresponds to the codon usage of the hostorganism intended for production of the enzyme, or by introduction ofnucleotide substitutions which may give rise to a different amino acidsequence. For a general description of nucleotide substitution, see,e.g., Ford et al., 1991, Protein Expression and Purification 2:95-107.

It will be apparent to those skilled in the art that such substitutionscan be made outside the regions critical to the function of the moleculeand still result in an active polypeptide. Amino acid residues essentialto the activity of the polypeptide encoded by the isolated nucleic acidsequence of the invention, and therefore preferably not subject tosubstitution, may be identified according to procedures known in theart, such as site-directed mutagenesis or alanine-scanning mutagenesis(see, e.g., Cunningham and Wells, 1989, Science 244:1081-1085). In thelatter technique mutations are introduced at every residue in themolecule, and the resultant mutant molecules are tested for mutanaseactivity to identify amino acid residues that are critical to theactivity of the molecule. Sites of substrate-enzyme interaction can alsobe determined by analysis of three-dimensional structure as determinedby such techniques as nuclear magnetic resonance analysis,crystallography or photoaffinity labelling (see, e.g., de Vos et al.,1992, Science 255, 306-312; Smith et al., 1992, Journal of MolecularBiology 224:899-904; Wlodaver et al., 1992, FEBS Letters 309, 59-64).

Polypeptides of the present invention also include fused polypeptides orcleavable fusion polypeptides in which another polypeptide is fused atthe N-terminus or the C-terminus of the polypeptide or fragment thereof.A fused polypeptide is produced by fusing a nucleic acid sequence (or aportion thereof) encoding another polypeptide to a nucleic acid sequence(or a portion thereof) of the present invention. Techniques forproducing fusion polypeptides are known in the art, and include,ligating the coding sequences encoding the polypeptides so that they arein frame and that expression of the fused polypeptide is under controlof the same promoter(s) and terminator.

The present invention also relates to nucleic acid sequences which arecapable of hybridizing under high stringency conditions with anoligonucleotide probe which hybridizes under the same conditions withthe nucleic acid sequence set forth in SEQ ID NO:2 or its complementarystrand (Sambrook et al., supra). Hybridization indicates that theanalogous nucleic acid sequence hybridizes to the oligonucleotide probecorresponding to the polypeptide encoding part of the nucleic acidsequence shown in SEQ ID NO:2 under standard conditions.

The amino acid sequence set forth in SEQ ID NO:3 or a partial amino acidsequence thereof may be used to design an oligonucleotide probe, or agene encoding a polypeptide of the present invention or a subsequencethereof can also be used as a probe, to isolate homologous genes of anygenus or species. In particular, such probes can be used forhybridization with the genomic or cDNA of the genus or species ofinterest, following standard Southern blotting procedures, in order toidentify and isolate the corresponding gene therein. Such probes can beconsiderably shorter than the entire sequence, but should be at least15, preferably at least 25, and more preferably at least 40 nucleotidesin length. Longer probes can also be used. Both DNA and RNA probes canbe used. The probes are typically labeled for detecting thecorresponding gene (for example, with ³² p, ³ H, ³⁵ S, biotin, oravidin). A PCR reaction using the degenerate probes mentioned herein andgenomic DNA or first-strand cDNA from a Penicillium purpurogenum straincan also yield a Penicillium purpurogenum mutanase-specific productwhich can then be used as a probe to clone the corresponding genomic orcDNA.

Nucleic Acid Constructs

The present invention also relates to nucleic acid constructs comprisinga nucleic acid sequence of the present invention operably linked to oneor more control sequences capable of directing the expression of thecoding sequence in a suitable host cell under conditions compatible withthe control sequences.

"Nucleic acid construct" is defined herein as a nucleic acid molecule,either single- or double-stranded, which is isolated from a naturallyoccurring gene or which has been modified to contain segments of nucleicacid which are combined and juxtaposed in a manner which would nototherwise exist in nature. The term nucleic acid construct may besynonymous with the term expression cassette when the nucleic acidconstruct contains all the control sequences required for expression ofa coding sequence of the present invention. The term "coding sequence"as defined herein is a sequence which is transcribed into mRNA andtranslated into a polypeptide of the present invention when placed underthe control of the above mentioned control sequences. The boundaries ofthe coding sequence are generally determined by a translation startcodon ATG at the 5'-terminus and a translation stop codon at the3'-terminus. A coding sequence can include, but is not limited to,genomic DNA, cDNA, and recombinant nucleic acid sequences.

An isolated nucleic acid sequence encoding a polypeptide of the presentinvention may be manipulated in a variety of ways to provide forexpression of the polypeptide. Manipulation of the nucleic acid sequenceencoding a polypeptide prior to its insertion into a vector may bedesirable or necessary depending on the expression vector. Thetechniques for modifying nucleic acid sequences utilizing cloningmethods are well known in the art.

The term "control sequences" is defined herein to include all componentswhich are necessary or advantageous for expression of the codingsequence of the nucleic acid sequence. Each control sequence may benative or foreign to the nucleic acid sequence encoding the polypeptide.Such control sequences include, but are not limited to, a leader, apolyadenylation sequence, a propeptide sequence, a promoter, a signalsequence, and a transcription terminator. At a minimum, the controlsequences include a promoter, and transcriptional and translational stopsignals. The control sequences may be provided with linkers for thepurpose of introducing specific restriction sites facilitating ligationof the control sequences with the coding region of the nucleic acidsequence encoding a polypeptide.

The control sequence may be an appropriate promoter sequence, a nucleicacid sequence which is recognized by a host cell for expression of thenucleic acid sequence. The promoter sequence contains transcriptioncontrol sequences which mediate the expression of the polypeptide. Thepromoter may be any nucleic acid sequence which shows transcriptionalactivity in the host cell of choice and may be obtained from genesencoding extracellular or intracellular polypeptides either homologousor heterologous to the host cell.

Examples of suitable promoters for directing the transcription of thenucleic acid constructs of the present invention, especially in abacterial host cell, are the promoters obtained from the E. coli lacoperon, the Streptomyces coelicolor agarase gene (dagA), the Bacillussubtilis levansucrase gene (sacB), the Bacillus licheniformisalpha-amylase gene (amyL), the Bacillus stearothermophilus maltogenicamylase gene (amyM), the Bacillus amyloliquefaciens alpha-amylase gene(amyQ), the Bacillus licheniformis penicillinase gene (penP), theBacillus subtilis xylA and xylB genes, and the prokaryoticbeta-lactamase gene (Villa-Kamaroff et al., 1978, Proceedings of theNational Academy of Sciences USA 75:3727-3731), as well as the tacpromoter (DeBoer et al., 1983, Proceedings of the National Academy ofSciences USA 80:21-25). Further promoters are described in "Usefulproteins from recombinant bacteria" in Scientific American, 1980,242:74-94; and in Sambrook et al., 1989, supra.

Examples of suitable promoters for directing the transcription of thenucleic acid constructs of the present invention in a filamentous fungalhost cell are promoters obtained from the genes encoding Aspergillusoryzae TAKA amylase, Rhizomucor miehei aspartic proteinase, Aspergillusniger neutral alpha-amylase, Aspergillus niger acid stablealpha-amylase, Aspergillus niger or Aspergillus awamori glucoamylase(glaA), Rhizomucor miehei lipase, Aspergillus oryzae alkaline protease,Aspergillus oryzae triose phosphate isomerase, Aspergillus nidulansacetamidase, Fusarium oxysporum trypsin-like protease (as described inU.S. Pat. No. 4,288,627, which is incorporated herein by reference), andhybrids thereof. Particularly preferred promoters for use in filamentousfungal host cells are the TAKA amylase, NA2-tpi (a hybrid of thepromoters from the genes encoding Aspergillus niger neutral a-amylaseand Aspergillus oryzae triose phosphate isomerase), and glaA promoters.

In a yeast host, useful promoters are obtained from the Saccharomycescerevisiae enolase (ENO-1) gene, the Saccharomyces cerevisiaegalactokinase gene (GAL1), the Saccharomyces cerevisiae alcoholdehydrogenase/glyceraldehyde-3-phosphate dehydrogenase genes (ADH2/GAP),and the Saccharomyces cerevisiae 3-phosphoglycerate kinase gene. Otheruseful promoters for yeast host cells are described by Romanos et al.,1992, Yeast 8:423-488. In a mammalian host cell, useful promotersinclude viral promoters such as those from Simian Virus 40 (SV40), Roussarcoma virus (RSV), adenovirus, and bovine papilloma virus (BPV).

The control sequence may also be a suitable transcription terminatorsequence, a sequence recognized by a host cell to terminatetranscription. The terminator sequence is operably linked to the 3'terminus of the nucleic acid sequence encoding the polypeptide. Anyterminator which is functional in the host cell of choice may be used inthe present invention.

Preferred terminators for filamentous fungal host cells are obtainedfrom the genes encoding Aspergillus oryzae TAKA amylase, Aspergillusniger glucoamylase, Aspergillus nidulans anthranilate synthase,Aspergillus niger alpha-glucosidase, and Fusarium oxysporum trypsin-likeprotease.

Preferred terminators for yeast host cells are obtained from the genesencoding Saccharomyces cerevisiae enolase, Saccharomyces cerevisiaecytochrome C (CYC1), or Saccharomyces cerevisiaeglyceraldehyde-3-phosphate dehydrogenase. Other useful terminators foryeast host cells are described by Romanos et al., 1992, supra.Terminator sequences are well known in the art for marnmalian hostcells.

The control sequence may also be a suitable leader sequence, anontranslated region of a mRNA which is important for translation by thehost cell. The leader sequence is operably linked to the 5' terminus ofthe nucleic acid sequence encoding the polypeptide. Any leader sequencewhich is functional in the host cell of choice may be used in thepresent invention.

Preferred leaders for filamentous fungal host cells are obtained fromthe genes encoding Aspergillus oryzae TAKA amylase and Aspergillusoryzae triose phosphate isomerase.

Suitable leaders for yeast host cells are obtained from theSaccharomyces cerevisiae enolase (ENO-1) gene, the Saccharomycescerevisiae 3-phosphoglycerate kinase gene, the Saccharomyces cerevisiaealpha-factor, and the Saccharomyces cerevisiae alcoholdehydrogenase/glyceraldehyde-3-phosphate dehydrogenase genes (ADH2/GAP).

The control sequence may also be a polyadenylation sequence, a sequencewhich is operably linked to the 3' terminus of the nucleic acid sequenceand which, when transcribed, is recognized by the host cell as a signalto add polyadenosine residues to transcribed mRNA. Any polyadenylationsequence which is functional in the host cell of choice may be used inthe present invention.

Preferred polyadenylation sequences for filamentous fungal host cellsare obtained from the genes encoding Aspergillus oryzae TAKA amylase,Aspergillus niger glucoamylase, Aspergillus nidulans anthranilatesynthase, and Aspergillus niger alpha-glucosidase.

Useful polyadenylation sequences for yeast host cells are described byGuo and Sherman, 1995, Molecular Cellular Biology 15:5983-5990.Polyadenylation sequences are well known in the art for mammalian hostcells.

The control sequence may also be a signal peptide coding region, whichcodes for an amino acid sequence linked to the amino terminus of thepolypeptide which can direct the expressed polypeptide into the cell'ssecretory pathway. The 5' end of the coding sequence of the nucleic acidsequence may inherently contain a signal peptide coding region naturallylinked in translation reading frame with the segment of the codingregion which encodes the secreted polypeptide. Alternatively, the 5' endof the coding sequence may contain a signal peptide coding region whichis foreign to that portion of the coding sequence which encodes thesecreted polypeptide. The foreign signal peptide coding region may berequired where the coding sequence does not normally contain a signalpeptide coding region. Alternatively, the foreign signal peptide codingregion may simply replace the natural signal peptide coding region inorder to obtain enhanced secretion of the mutanase relative to thenatural signal is peptide coding region normally associated with thecoding sequence. The signal peptide coding region may be obtained from aglucoamylase or an amylase gene from an Aspergillus species, a lipase orproteinase gene from a Rhizomucor species, the gene for the alpha-factorfrom Saccharomyces cerevisiae , an amylase or a protease gene from aBacillus species, or the calf preprochymosin gene. However, any signalpeptide coding region capable of directing the expressed mutanase intothe secretory pathway of a host cell of choice may be used in thepresent invention.

An effective signal peptide coding region for bacterial host cells isthe signal peptide coding region obtained from the maltogenic amylasegene from Bacillus NCIB 11837, the Bacillus stearothermophilusalpha-amylase gene, the Bacillus licheniformis subtilisin gene, theBacillus licheniformis beta-lactamase gene, the Bacillusstearothermophilus neutral proteases genes (nprT, nprS, nprM), and theBacillus subtilis PrsA gene. Further signal peptides are described bySimonen and Palva, 1993, Microbiological Reviews 57:109-137.

An effective signal peptide coding region for filamentous fungal hostcells is the signal peptide coding region obtained from Aspergillusoryzae TAKA amylase gene, Aspergillus niger neutral amylase gene, theRhizomucor miehei aspartic proteinase gene, the Humicola lanuginosacellulase gene, or the Rhizomucor miehei lipase gene.

Useful signal peptides for yeast host cells are obtained from the genesfor Saccharomyces cerevisiae a-factor and Saccharomyces cerevisiaeinvertase. Other useful signal peptide coding regions are described byRomanos et al., 1992, supra.

The control sequence may also be a propeptide coding region, which codesfor an amino acid sequence positioned at the amino terminus of apolypeptide. The resultant polypeptide is known as a proenzyme orpropolypeptide (or a zymogen in some cases). A propolypeptide isgenerally inactive and can be converted to mature active polypeptide bycatalytic or autocatalytic cleavage of the propeptide from thepropolypeptide. The propeptide coding region may be obtained from theBacillus subtilis alkaline protease gene (aprE), the Bacillus subtilisneutral protease gene (nprT), the Saccharomyces cerevisiae alpha-factorgene, or the Myceliophthora thermophilum laccase gene (WO 95/33836).

The nucleic acid constructs of the present invention may also compriseone or more nucleic acid sequences which encode one or more factors thatare advantageous in the expression of the polypeptide, e.g., anactivator (e.g., a trans-acting factor), a chaperone, and a processingprotease. Any factor that is functional in the host cell of choice maybe used in the present invention. The nucleic acids encoding one or moreof these factors are not necessarily in tandem with the nucleic acidsequence encoding the polypeptide.

An activator is a protein which activates transcription of a nucleicacid sequence encoding a polypeptide (Kudla et al., 1990, EMBO Journal9:1355-1364; Jarai and Buxton, 1994, Current Genetics 26:2238-244;Verdier, 1990, Yeast 6:271-297). The nucleic acid sequence encoding anactivator may be obtained from the genes encoding Bacillusstearothermophilus NprA (nprA), Saccharomyces cerevisiae heme activatorprotein 1 (hap1), Saccharomyces cerevisiae galactose metabolizingprotein 4 (gal4), and Aspergillus nidulans ammonia regulation protein(areA). For further examples, see Verdier, 1990, supra and MacKenzie etal., 1993, Journal of General Microbiology 139:2295-2307.

A chaperone is a protein which assists another polypeptide in foldingproperly (Hartl et al., 1994, TIBS 19:20-25; Bergeron et al., 1994, TIBS19:124-128; Demolder et al., 1994, Journal of Biotechnology 32:179-189;Craig, 1993, Science 260:1902-1903; Gething and Sambrook, 1992, Nature355:33-45; Puig and Gilbert, 1994, Journal of Biological Chemistry269:7764-7771; Wang and Tsou, 1993, The FASEB Journal 7:1515-11157;Robinson et al., 1994, Bio/Technology 1:381-384). The nucleic acidsequence encoding a chaperone may be obtained from the genes encodingBacillus subtilis GroE proteins, Aspergillus oryzae protein disulphideisomerase, Saccharomyces cerevisiae calnexin, Saccharomyces cerevisiaeBiP/GRP78, and Saccharomyces cerevisiae Hsp7O. For further examples, seeGething and Sambrook, 1992, supra, and Hartl et al., 1994, supra.

A processing protease is a protease that cleaves a propeptide togenerate a mature biochemically active polypeptide (Enderlin andOgrydziak, 1994, Yeast 10:67-79; Fuller et al., 1989, Proceedings of theNational Academy of Sciences USA 86:1434-1438; Julius et al., 1984, Cell37:1075-1089; Julius et al., 1983, Cell 32:839-852). The nucleic acidsequence encoding a processing protease may be obtained from the genesencoding Saccharomyces cerevisiae dipeptidylaminopeptidase,Saccharomyces cerevisiae Kex2, and Yarrowia lipolytica dibasicprocessing endoprotease (xpr6).

It may also be desirable to add regulatory sequences which allow theregulation of the expression of the polypeptide relative to the growthof the host cell. Examples of regulatory systems are those which causethe expression of the gene to be turned on or off in response to achemical or physical stimulus, including the presence of a regulatorycompound.

Regulatory systems in prokaryotic systems would include the lac, tac,and trp operator systems. In yeast, the ADH2 system or GAL1 system maybe used. In filamentous fungi, the TAKA alpha-amylase promoter,Aspergillus niger glucoamylase promoter, and the Aspergillus oryzaeglucoamylase promoter may be used as regulatory sequences. Otherexamples of regulatory sequences are those which allow for geneamplification. In eukaryotic systems, these include the dihydrofolatereductase gene which is amplified in the presence of methotrexate, andthe metallothionein genes which are amplified with heavy metals. Inthese cases, the nucleic acid sequence encoding the polypeptide would beplaced in tandem with the regulatory sequence.

Expression Vectors

The present invention also relates to recombinant expression vectorscomprising a nucleic acid sequence of the present invention, a promoter,and transcriptional and translational stop signals. The various nucleicacid and control sequences described above may be joined together toproduce a recombinant expression vector which may include one or moreconvenient restriction sites to allow for insertion or substitution ofthe nucleic acid sequence encoding the polypeptide at such sites.Alternatively, the nucleic acid sequence of the present invention may beexpressed by inserting the nucleic acid sequence or a nucleic acidconstruct comprising the sequence into an appropriate vector forexpression. In creating the expression vector, the coding sequence islocated in the vector so that the coding sequence is operably linkedwith the appropriate control sequences for expression, and possiblysecretion.

The recombinant expression vector may be any vector (e.g., a plasmid orvirus) which can be conveniently subjected to recombinant DNA proceduresand can bring about the expression of the nucleic acid sequence. Thechoice of the vector will typically depend on the compatibility of thevector with the host cell into which the vector is to be introduced. Thevectors may be linear or closed circular plasmids. The vector may be anautonomously replicating vector, i.e., a vector which exists as anextrachromosomal entity, the replication of which is independent ofchromosomal replication, e.g., a plasmid, an extrachromosomal element, aminichromosome, or an artificial chromosome. The vector may contain anymeans for assuring self-replication. Alternatively, the vector may beone which, when introduced into the host cell, is integrated into thegenome and replicated together with the chromosome(s) into which it hasbeen integrated. The vector system may be a single vector or plasmid ortwo or more vectors or plasmids which together contain the total DNA tobe introduced into the genome of the host cell, or a transposon.

The vectors of the present invention preferably contain one or moreselectable markers which permit easy selection of transformed cells. Aselectable marker is a gene the product of which provides for biocide orviral resistance, resistance to heavy metals, prototrophy to auxotrophs,and the like. Examples of bacterial selectable markers are the dal genesfrom Bacillus subtilis or Bacillus licheniformis, or markers whichconfer antibiotic resistance such as ampicillin, kanamycin,chloramphenicol or tetracycline resistance. A frequently used mammalianmarker is the dihydrofolate reductase gene. Suitable markers for yeasthost cells are ADE2, HIS3, LEU2, LYS2, MET3, TRP1, and URA3. Aselectable marker for use in a filamentous fungal host cell may beselected from the group including, but not limited to, amdS(acetamidase), argB (ornithine carbamoyltransferase), bar(phosphinothricin acetyltransferase), hygB (hygromycinphosphotransferase), niaD (nitrate reductase), pyrG (orotidine-5'-phosphate decarboxylase), sC (sulfate adenyltransferase), trpC(anthranilate synthase), and glufosinate resistance markers, as well asequivalents from other species. Preferred for use in an Aspergillus cellare the amdS and pyrG markers of Aspergillus nidulans or Aspergillusoryzae and the bar marker of Streptomyces hygroscopicus. Furthermore,selection may be accomplished by co-transformation, e. g., as describedin WO 91/17243, where the selectable marker is on a separate vector.

The vectors of the present invention preferably contain an element(s)that permits stable integration of the vector into the host cell genomeor autonomous replication of the vector in the cell independent of thegenome of the cell.

The vectors of the present invention may be integrated into the hostcell genome when introduced into a host cell. For integration, thevector may rely on the nucleic acid sequence encoding the polypeptide orany other element of the vector for stable integration of the vectorinto the genome by homologous or nonhomologous recombination.Alternatively, the vector may contain additional nucleic acid sequencesfor directing integration by homologous recombination into the genome ofthe host cell. The additional nucleic acid sequences enable the vectorto be integrated into the host cell genome at a precise location(s) inthe chromosome(s). To increase the likelihood of integration at aprecise location, the integrational elements should preferably contain asufficient number of nucleic acids, such as 100 to 1,500 base pairs,preferably 400 to 1,500 base pairs, and most preferably 800 to 1,500base pairs, which are highly homologous with the corresponding targetsequence to enhance the probability of homologous recombination. Theintegrational elements may be any sequence that is homologous with thetarget sequence in the genome of the host cell. Furthermore, theintegrational elements may be non-encoding or encoding nucleic acidsequences. On the other hand, the vector may be integrated into thegenome of the host cell by non-homologous recombination. These nucleicacid sequences may be any sequence that is homologous with a targetsequence in the genome of the host cell, and, furthermore, may benon-encoding or encoding sequences.

For autonomous replication, the vector may further comprise an origin ofreplication enabling the vector to replicate autonomously in the hostcell in question. Examples of bacterial origins of replication are theorigins of replication of plasmids pBR322, pUC19, pACYC177, and pACYC184permitting replication in E. coli, and pUB110, pE194, pTA1060, and pAMB1permitting replication in Bacillus. Examples of origin of replicationsfor use in a yeast host cell are the 2 micron origin of replication, thecombination of CEN6 and ARS4, and the combination of CEN3 and ARS1. Theorigin of replication may be one having a mutation which makes itsfunctioning temperature-sensitive in the host cell (see, e.g., Ehrlich,1978, Proceedings of the National Academy of Sciences USA 75:1433).

More than one copy of a nucleic acid sequence encoding a polypeptide ofthe present invention may be inserted into the host cell to amplifyexpression of the nucleic acid sequence. Stable amplification of thenucleic acid sequence can be obtained by integrating at least oneadditional copy of the sequence into the host cell genome using methodswell known in the art and selecting for transformants.

The procedures used to ligate the elements described above to constructthe recombinant expression vectors of the present invention are wellknown to one skilled in the art (see, e.g., Sambrook et al., 1989,supra).

Host Cells

The present invention also relates to recombinant host cells, comprisinga nucleic acid sequence of the invention, which are advantageously usedin the recombinant production of the polypeptides. The term "host cell"encompasses any progeny of a parent cell which is not identical to theparent cell due to mutations that occur during replication.

The cell is preferably transformed with a vector comprising a nucleicacid sequence of the invention followed by integration of the vectorinto the host chromosome. "Transformation" means introducing a vectorcomprising a nucleic acid sequence of the present invention into a hostcell so that the vector is maintained as a chromosomal integrant or as aself-replicating extra-chromosomal vector. Integration is generallyconsidered to be an advantage as the nucleic acid sequence is morelikely to be stably maintained in the cell. Integration of the vectorinto the host chromosome may occur by homologous or non-homologousrecombination as described above.

The choice of a host cell will to a large extent depend upon the geneencoding the polypeptide and its source. The host cell may be aunicellular microorganism, e.g., a prokaryote, or a non-unicellularmicroorganism, e.g., a eukaryote. Useful unicellular cells are bacterialcells such as gram positive bacteria including, but not limited to, aBacillus cell, e.g., Bacillus alkalophilus, Bacillus amyloliquefaciens,Bacillus brevis, Bacillus circulans, Bacillus coagulans, Bacilluslautus, Bacillus lentus, Bacillus licheniformis, Bacillus megaterium,Bacillus stearothermophilus, Bacillus subtilis, and Bacillusthuringiensis; or a Streptomyces cell, e.g., Streptomyces lividans orStreptomyces murinus, or gram negative bacteria such as E. coli andPseudomonas sp. In a preferred embodiment, the bacterial host cell is aBacillus lentus, Bacillus licheniformis, Bacillus stearothermophilus orBacillus subtilis cell. The transformation of a bacterial host cell may,for instance, be effected by protoplast transformation (see, e.g., Changand Cohen, 1979, Molecular General Genetics 168:111-115), by usingcompetent cells (see, e.g., Young and Spizizin, 1961, Journal ofBacteriology 81:823-829, or Dubnar and Davidoff-Abelson, 1971, Journalof Molecular Biology 56:209-221), by electroporation (see, e.g.,Shigekawa and Dower, 1988, Biotechniques 6:742-751), or by conjugation(see, e.g., Koehler and Thorne, 1987, Journal of Bacteriology169:5771-5278).

The host cell may be a eukaryote, such as a mammalian cell, an insectcell, a plant cell or a fungal cell. Useful mammalian cells includeChinese hamster ovary (CHO) cells, HeLa cells, baby hamster kidney (BHK)cells, COS cells, or any number of other immortalized cell linesavailable, e.g., from the American Type Culture Collection.

In a preferred embodiment, the host cell is a fungal cell. "Fungi" asused herein includes the phyla Ascomycota, Basidiomycota,Chytridiomycota, and Zygomycota (as defined by Hawksworth et al., In,Ainsworth and Bisby's Dictionary of The Fungi, 8th edition, 1995, CABInternational, University Press, Cambridge, UK) as well as the Oomycota(as cited in Hawksworth et al., 1995, supra, page 171) and allmitosporic fungi (Hawksworth et al., 1995, supra). Representative groupsof Ascomycota include, e.g., Neurospora, Eupenicillium (=Penicillium),Emericella (=Aspergillus), Eurotium (=Aspergillus), and the true yeastslisted above. Examples of Basidiomycota include mushrooms, rusts, andsmuts. Representative groups of Chytridiomycota include, e.g.,Allomyces, Blastocladiella, Coelomomyces, and aquatic fungi.Representative groups of Oomycota include, e.g., Saprolegniomycetousaquatic fungi (water molds) such as Achlya. Examples of mitosporic fungiinclude Aspergillus, Penicillium, Candida, and Alternaria.Representative groups of Zygomycota include, e.g., Rhizopus and Mucor.

In a preferred embodiment, the fungal host cell is a yeast cell. "Yeast"as used herein includes ascosporogenous yeast (Endomycetales),basidiosporogenous yeast, and yeast belonging to the Fungi Imperfecti(Blastomycetes). The ascosporogenous yeasts are divided into thefamilies Spermophthoraceae and Saccharomycetaceae. The latter iscomprised of four subfamilies, Schizosaccharomycoideae (e. g., genusSchizosaccharomyces), Nadsonioideae, Lipomycoideae, andSaccharomycoideae (e.g., genera Pichia, Kluyveromyces andSaccharomyces). The basidiosporogenous yeasts include the generaLeucosporidim, Rhodosporidium, Sporidiobolus, Filobasidium, andFilobasidiella. Yeast belonging to the Fungi Imperfecti are divided intotwo families, Sporobolomycetaceae (e.g., genera Sorobolomyces andBullera) and Cryptococcaceae (e.g., genus Candida). Since theclassification of yeast may change in the future, for the purposes ofthis invention, yeast shall be defined as described in Biology andActivities of Yeast (Skinner, F. A., Passmore, S. M., and Davenport, R.R., eds, Soc. App. Bacteriol. Symposium Series No. 9, 1980. The biologyof yeast and manipulation of yeast genetics are well known in the art(see, e.g., Biochemistry and Genetics of Yeast, Bacil, M., Horecker, B.J., and Stopani, A. O. M., editors, 2nd edition, 1987; The Yeasts, Rose,A. H., and Harrison, J. S., editors, 2nd edition, 1987; and TheMolecular Biology of the Yeast Saccharomyces, Strathern et al., editors,1981).

In a more preferred embodiment, the yeast host cell is a cell of aspecies of Candida, Kluyveromyces, Saccharomyces, Schizosaccharomyces,Pichia, or Yarrowia.

In a most preferred embodiment, the yeast host cell is a Saccharomycescarlsbergensis, Saccharomyces cerevisiae, Saccharomyces diastaticus,Saccharomyces douglasii, Saccharomyces kluyveri, Saccharomyces norbensisor Saccharomyces oviformis cell. In another most preferred embodiment,the yeast host cell is a Kluyveromyces lactis cell. In another mostpreferred embodiment, the yeast host cell is a Yarrowia lipolytica cell.

In a preferred embodiment, the fungal host cell is a filamentous fungalcell. "Filamentous fungi" include all filamentous forms of thesubdivision Eumycota and Oomycota (as defined by Hawksworth et al.,1995, supra). The filamentous fungi are characterized by a vegetativemycelium composed of chitin, cellulose, glucan, chitosan, mannan, andother complex polysaccharides. Vegetative growth is by hyphal elongationand carbon catabolism is obligately aerobic. In contrast, vegetativegrowth by yeasts such as Saccharomyces cerevisiae is by budding of aunicellular thallus and carbon catabolism may be fermentative. In a morepreferred embodiment, the filamentous fungal host cell is a cell of aspecies of, but not limited to, Acremonium, Aspergillus, Fusarium,Humicola, Mucor, Myceliophthora, Neurospora, Penicillium, Thielavia,Tolypocladium, and Trichoderma.

In an even more preferred embodiment, the filamentous fungal host cellis an Aspergillus cell. In another even more preferred embodiment, thefilamentous fungal host cell is an Acremonium cell. In another even morepreferred embodiment, the filamentous fungal host cell is a Fusariumcell. In another even more preferred embodiment, the filamentous fungalhost cell is a Humicola cell. In another even more preferred embodiment,the filamentous fungal host cell is a Mucor cell. In another even morepreferred embodiment, the filamentous fungal host cell is aMyceliophthora cell. In another even more preferred embodiment, thefilamentous fungal host cell is a Neurospora cell. In another even morepreferred embodiment, the filamentous fungal host cell is a Penicilliumcell. In another even more preferred embodiment, the filamentous fungalhost cell is a Thielavia cell. In another even more preferredembodiment, the filamentous fungal host cell is a Tolypocladium cell. Inanother even more preferred embodiment, the filamentous fungal host cellis a Trichoderma cell.

In a most preferred embodiment, the filamentous fungal host cell is anAspergillus awamori, Aspergillus foetidus, Aspergillus japonicus,Aspergillus niger or Aspergillus oryzae cell. In another most preferredembodiment, the filamentous fungal host cell is a Fusarium cerealis,Fusarium crookwellense, Fusarium graminearum, Fusarium oxysporum,Fusarium sambucinum or Fusarium sulphureum cell. In another mostpreferred embodiment, the filamentous fungal host cell is a Humicolainsolens or Humicola lanuginosa cell. In another most preferredembodiment, the filamentous fungal host cell is a Mucor miehei cell. Inanother most preferred embodiment, the filamentous fungal host cell is aMyceliophthora thermophilum cell. In another most preferred embodiment,the filamentous fungal host cell is a Neurospora crassa cell. In anothermost preferred embodiment, the filamentous fungal host cell is aPenicillium purpurogenum cell. In another most preferred embodiment, thefilamentous fungal host cell is a Thielavia terrestris cell. In anothermost preferred embodiment, the Trichoderma cell is a Trichodermaharzianum, Trichoderma koningii, Trichoderma longibrachiatum,Trichoderma reesei or Trichoderma viride cell.

Fungal cells may be transformed by a process involving protoplastformation, transformation of the protoplasts, and regeneration of thecell wall in a manner known per se. Suitable procedures fortransformation of Aspergillus host cells are described in EP 238 023 andYelton et al., 1984, Proceedings of the National Academy of Sciences USA81:1470-1474. A suitable method of transforming Fusarium species isdescribed by Malardier et al., 1989, Gene 78:147-156 or in copendingU.S. Ser. No. 08/269,449. Yeast may be transformed using the proceduresdescribed by Becker and Guarente, In Abelson, J. N. and Simon, M. I.,editors, Guide to Yeast Genetics and Molecular Biology, Methods inEnzymology, Volume 194, pp 182-187, Academic Press, Inc., New York; Itoet al., 1983, Journal of Bacteriology 153:163; and Hinnen et al., 1978,Proceedings of the National Academy of Sciences USA 75:1920. Mammaliancells may be transformed by direct uptake using the calcium phosphateprecipitation method of Graham and Van der Eb (1978, Virology 52:546).

Methods of Production

The present invention also relates to methods for producing apolypeptide of the present invention comprising (a) cultivating aPenicillium strain to produce a supernatant comprising the polypeptide;and (b) recovering the polypeptide.

The present invention also relates to methods for producing apolypeptide of the present invention comprising (a) cultivating a hostcell under conditions conducive to expression of the polypeptide; and(b) recovering the polypeptide.

In both methods, the cells are cultivated in a nutrient medium suitablefor production of the polypeptide using methods known in the art. Forexample, the cell may be cultivated by shake flask cultivation,small-scale or large-scale fermentation (including continuous, batch,fed-batch, or solid state fermentations) in laboratory or industrialfermentors performed in a suitable medium and under conditions allowingthe polypeptide to be expressed and/or isolated. The cultivation takesplace in a suitable nutrient medium comprising carbon and nitrogensources and inorganic salts, using procedures known in the art (see,e.g., references for bacteria and yeast; Bennett, J. W. and LaSure, L.,editors, More Gene Manipulations in Fungi, Academic Press, Calif.,1991). Suitable media are available from commercial suppliers or may beprepared according to published compositions (e.g., in catalogues of theAmerican Type Culture Collection). If the polypeptide is secreted intothe nutrient medium, the polypeptide can be recovered directly from themedium. If the polypeptide is not secreted, it is recovered from celllysates.

The polypeptides may be detected using methods known in the art that arespecific for the polypeptides. These detection methods may include useof specific antibodies, formation of an enzyme product, or disappearanceof an enzyme substrate. For example, an enzyme assay may be used todetermine the activity of the polypeptide. Procedures for determiningmutanase activity are known in the art and include, e.g., highperformance size exclusion chromatography of mutanase-digested mutan.

The resulting polypeptide may be recovered by methods known in the art.For example, the polypeptide may be recovered from the nutrient mediumby conventional procedures including, but not limited to,centrifugation, filtration, extraction, spray-drying, evaporation, orprecipitation. The recovered polypeptide may then be further purified bya variety of chromatographic procedures, e.g., ion exchangechromatography, gel filtration chromatography, affinity chromatography,or the like.

The polypeptides of the present invention may be purified by a varietyof procedures known in the art including, but not limited to,chromatography (e.g., ion exchange, affinity, hydrophobic,chromatofocusing, and size exclusion), electrophoretic procedures (e.g.,preparative isoelectric focusing (IEF), differential solubility (e.g.,ammonium sulfate precipitation), or extraction (see, e.g., ProteinPurification, J.-C. Janson and Lars Ryden, editors, VCH Publishers, NewYork, 1989).

Polypeptide Compositions

In a still further aspect, the present invention relates to polypeptidecompositions which are enriched in a polypeptide of the presentinvention. In the present context, the term "enriched" is intended toindicate that the mutanase activity of the polypeptide composition hasbeen increased, e.g., with an enrichment factor of 1.1, conveniently dueto addition of a polypeptide of the invention.

The polypeptide composition may be one which comprises a polypeptide ofthe invention as the major enzymatic component, e.g., a mono-componentpolypeptide composition. Alternatively, the composition may comprisemultiple enzymatic activities, such as an aminopeptidase, an amylase, acarbohydrase, a carboxypeptidase, a catalase, a cellulase, a chitinase,a cutinase, a deoxyribonuclease, an esterase, an alpha-galactosidase, abeta-galactosidase, a glucoamylase, an alpha-glucosidase, abeta-glucosidase, a haloperoxidase, an invertase, a laccase, a lipase, amannosidase, a mutanase, an oxidase, a pectinolytic enzyme, aperoxidase, a phytase, a polyphenoloxidase, a proteolytic enzyme, aribonuclease, or a xylanase. The additional enzyme(s) may be producibleby means of a microorganism belonging to the genus Aspergillus,preferably Aspergillus aculeatus, Aspergillus awamori, Aspergillusniger, or Aspergillus oryzae, or Trichoderina, Humicola, preferablyHumicola insolens, or Fusarium, preferably Fusarium graminearum.

The polypeptide compositions may be prepared in accordance with methodsknown in the art and may be in the form of a liquid or a drycomposition. For instance, the polypeptide composition may be in theform of a granulate or a microgranulate. The polypeptide to be includedin the composition may be stabilized in accordance with methods known inthe art.

Examples are given below of preferred uses of the polypeptidecompositions of the invention. The dosage of the polypeptide compositionof the invention and other conditions under which the composition isused may be determined on the basis of methods known in the art.

Uses

The mutanase of the present invention can be used as an antiplaque agentto degrade mutan produced by Streptococcus mutans in the oral cavity(Guggenheim, 1970, Helv. Odont. Acta 14:89-108). Mutan plays animportant role for adhesion and proliferation of bacteria on the surfaceof teeth and, hence, may be important in the etiology of dental caries(Kelstrup, 1978, Danish Dental Journal 82:431-437).

The present invention is also directed to oral cavity compositions,particularly dentifrices, comprising the mutanase in an effective amountand a suitable oral carrier for use as an antiplaque agent in dentalapplications and personal care products. "Effective amount" is definedherein as a sufficient amount of the mutanase to reduce plaque."Suitable oral carrier" is defined herein as a suitable vehicle whichcan be used to apply the compositions of the present invention to theoral cavity in a safe and effective manner. The compositions of thepresent invention can be made using methods which are common in the oralproduct area. Dentifrices are compositions used in conjunction with atoothbrush to remove stains from teeth and to leave the mouth feelingclean and refreshed after brushing. Dentifrices are also used to deliveragents with specific therapeutic and cosmetic functions. Examples ofpersonal care products include, but are not limited to, toothpaste,toothgel, mouthwash, chewing gum, and denture cleaners.

The composition ingredients will vary depending on the particularproduct (Kirk-Othmer, John Wiley & Son, New York). Examples ofingredients include, but are not limited to, an abrasive, a humectant, asurfactant, an emulsifier, a colloid, a chelating agent, an adhesive,one or more gums or resins for cohesiveness and structure, one or moreflavor agents, color, a preservative and active agents for specificeffects (e.g., fluoride and whiteners). Mouthwashes can deliver activeagents that cannot be provided by toothpaste because of chemicalincompatibilty between the agent and the toothpaste ingredients. Forexample, sodium fluoride, calcium-containing abrasives, sodium laurylsulfate, and chlorhexidine are incompatible.

The present invention is also directed to a method for degrading mutanin an oral cavity comprising applying to the oral cavity an effectiveamount of the compositions of the present invention. The compositions ofthe present invention can be applied in a dry, paste, gum, or liquidform. The composition may be a concentrate which requires dilution witha suitable quantity of water or other diluent before application. Theconcentrations of each component in the composition will vary dependingon the use and method of application. The mutanase concentration willvary depending upon the nature of the particular composition,specifically, whether it is a concentrate or to be used directly. Afterapplication, the composition is then allowed to remain in contact withthe tissues of the oral cavity for a period of time ranging from about15 seconds to about 12 hours until removed by rinsing or brushing.Alternatively, the composition may be left indefinitely until thecomposition is removed by a mechanical process, e.g., drinking liquid orchewing food.

The present invention is further described by the following exampleswhich should not be construed as limiting the scope of the invention.

EXAMPLES Example 1

Production of Mutanase by Penicillium purpurogenum CBS 238.95

Penicillium purpurogenum CBS 238.95 was obtained from the Centraalbureauvoor Schimmelcultures, Oosterstraat 1, 3742 SK Baarn, The Netherlands.The strain was cultivated at pH 6.0, 30° C., and 300 rpm in a mediumcomprised of 30 g of glucose, 0.5 g of yeast extract, 2 g of citricacid, 11 g of MgSO₄ -7H₂ O, 6 g of K₃ PO₄ -3H₂ O, 12 g of (NH₄)₂ HPO₄,and 6.5 g of lactic acid per liter. After 10 days of growth, the wholeculture broth was centrifuged and the supernatant recovered.

Example 2

Mutanase Plate Assay

Mutanase activity was detected by the ability of a sample of thesupernatant of Example 1 to produce clearing zones in mutan agar plates.The sensitivity of the plate assay was increased if the mutan wastreated with dextranase.

The dextranase-treated mutan was prepared by growing Streptococcusmutans CBS 350.71 at pH 6.5, 37° C. (kept constant), and with anaeration rate of 75 rpm in a medium comprised of 6.5 g of NZ-Case, 6 gof yeast extract, 20 g of (NH₄)₂ SO₄, 3 g of K₂ PO₄, 50 g of glucose,and 0.1% Pluronic PE6100 per liter.

After 35 hours, sucrose was added to a final concentration of 60 g/literto induce production of glucosyltransferase. The total fermentation timewas 75 hours. The supernatant from the fermentation was centrifuged andfiltered (sterile). Sucrose was then added to the supernatant to a finalconcentration of 5% (pH was adjusted to pH 7.0 with acetic acid) and thesolution was stirred overnight at 37° C. The solution was filtered andthe insoluble mutan was harvested on propex and washed extensively withdeionized water containing 1% sodium benzoate, pH 5 (adjusted withacetic acid). Finally, the insoluble mutan was lyophilized and ground.

Ten grams of purified Streptococcus mutans mutan was suspended in 200 mlof 0.1 M sodium acetate pH 6.0 and incubated at 30° C. for 20 hours with50 μl of DEXTRANASE™ 50 L (Novo Nordisk A/S, Bagsvaerd, Denmark).Following incubation, the suspension was centrifuged and the sedimentwas washed with deionized water. This step was repeated two times. Thewashed sediment was dried at 65° C. and ground into a powder using acoffee mill. A 1 gm quantity of the dextranase-treated mutan wassuspended in 15 ml of 0.1M sodium acetate pH 6.0 and blended for 25minutes in an Ultra Turrax homogenizer (Janke & Kunkel,IKA-Labortechnik). The blended suspension was autoclaved for 20 minutes,added to 450 ml of 2% molten agar, and poured into Petri plates. Aftercooling of the mutan-containing agar solution, wells were punched intothe agar and enzyme samples of 10 μl were placed in the wells. Theplates were incubated for 20 hours at 37° C. and mutanase activity wasvisualized as clear zones on a milky white background.

A 10 μl sample of the supernatant of the whole broth of Penicilliumpurpurogenum CBS 238.95 prepared as described in Example 1 produced aclearing zone on agar plates containing dextranase-treated mutan.

Example 3

High Performance Size Exclusion Chromatography Assay

The degradation of dextranase-treated mutan to soluble saccharideproducts by mutanase was determined by high performance size exclusionchromatography.

A 0.5% w/v suspension of dextranase-treated mutan (prepared as describedin Example 2) in 0.1M sodium acetate pH 6.0 was blended in an UltraTurrax homogenizer for 25 minutes. In an Eppendorf tube, 1 ml of theblended suspension was added to 20 μl of enzyme sample and incubated for20 hours at 30° C. in an Eppendorf thermomixer followed by heatinactivation of the mutanase at 95° C. for 20 minutes. For each mutanasesample, a control was run in which the mutanase solution was firstinactivated. The mutan suspensions were centrifuged and the supernatantswere analyzed by injecting 25 μl onto three TSK columns--PW G4000, PWG3000, PW G2500--(Toso Haas, 7.8 mm I.D.×30 cm) connected in tandem. Thesaccharides were eluted with 0.4M sodium acetate pH 3.0 at a temperatureof room temperature and a flow rate of 0.8 ml per minute. Elutingsaccharides were detected by refractive index using a Shimadzurefractive index detector and the data collected was processed usingDionex software (AI-450, Dionex Corporation, Sunnyvale, Calif.).Dextrans and glucose were used as molecular weight standards. Mutanaseactivity results in the production of glucose.

A 25 μl sample of the supernatant of the whole broth of Penicilliumpurpurogenum CBS 238.95 prepared as described in Example 1 producesglucose from dextranase-treated mutan.

Example 4

Purification of Penicillium purpurogenum CBS 238.95 Mutanase

The Penicillium purpurogenum CBS 238.95 mutanase was purified from thewhole broth supernatant prepared as described in Example 1 using afour-step purification method.

First, the supernatant was filtered through a 0.2 μm filter. Then 100 mlof the filtered supernatant was concentrated and equilibrated in 25 mMTris-HCI pH 8.0 by ultrafiltration using an Amicon cell equipped with a10,000 kDa MW-CO (molecular weight cut-off) membrane.

Second, the 50 ml concentrate was loaded at a flow rate of 1.5 ml perminute onto a XK 16/20 Fast Flow Q Sepharose (Pharmacia Biotech,Uppsala, Sweden) anion exchange column pre-equilibrated with 25 mMTris-HCI pH 8.0. The column was then washed with two volumes of 25 mMTris-HCI pH 8.0 before the bound proteins were eluted with a lineargradient from 0 to 1M NaCl in 25 mM Tris-HCl pH 8.0 in 3 column volumes.The fractions were assayed for mutanase activity using mutan agar platesas described in Example 2. The presence of mutanase activity wasconfirmed using the high performance size exclusion chromatographymethod described in Example 3. Fractions containing mutanase activitywere pooled. Mutanase activity eluted at approximately 0.75M NaCl.

Third, the buffer in the pooled fractions was changed to 0.25M ammoniumacetate pH 5.5 by equilibration by ultrafiltration using an Amicon cellequipped with a 10,000 kDa MW-CO membrane. The pooled fractions werethen loaded onto a HiLoad 26/60 Superdex 75 (Pharmacia Biotech, Uppsala,Sweden) gel filtration column and the mutanase protein was eluted at 1ml per minute with 0.25M ammonium acetate pH 5.5. The presence ofmutanase activity was determined using the high performance sizeexclusion chromatography method described in Example 3. Fractionscontaining mutanase activity were pooled.

Fourth, the buffer in the pooled fractions was changed to 20 mM Tris-HCIpH 8.0 by ultrafiltration using an Amicon cell equipped with a 10,000kDa MW-CO membrane. The pooled fractions were loaded at 1 ml per minuteonto a Mono Q HR10/10 (Pharmacia Biotech, Uppsala, Sweden) columnpre-equilibrated with 20 mM Tris-HCI pH 8.0. The column was then washedwith two volumes of 20 mM Tris-HCI pH 8.0 before the bound proteins wereeluted with a 100 ml linear gradient from 0 to 0.75M NaCi in 20 mMTris-HCI pH 8.0. Mutanase activity was determined using the highperformance size exclusion chromatography method described in Example 3.Mutanase activity eluted at approximately 0.4M NaCl.

Example 5

N-Terminal Sequencing of the Penicillium purpurogenum CBS 238.95Mutanase

N-terminal amino acid sequencing of the mutanase obtained fromPenicillium purpurogenum CBS 238.95 was performed following SDS-PAGE andelectroblotting using standard procedures with an Applied Biosystems473A protein sequencer equipped with a blot cartridge and operatedaccording to the manufacturer's instructions. The N-terminal amino acidsequence was determined to be as follows: ##STR1## wherein the aminoacid residues at positions 10 and 12 are uncertain, but are believed tobe His and Met, respectively, Xaa at position 1 designates anunidentified amino acid residue, and Asx at position 4 denotes an aminoacid residue which is either Asp or Asn. This sequence is clearlydistinct from the N-terminal sequence of the Trichoderma harzianummutanase disclosed in Japanese Patent No. 4-58889/A shown below:##STR2##

Example 6

Penicillium purpurogenum CBS 238.95 DNA Extraction

Penicillium purpurogenum CBS 238.95 was grown in 25 ml of 0.5% yeastextract-2% glucose (YEG) medium for 24 hours at 32° C. and 250 rpm.Mycelia were then collected by filtration through Miracloth (Calbiochem,La Jolla, Calif.) and washed once with 25 ml of 10 mM Tris-1 mM EDTA(TE) buffer. Excess buffer was drained from the mycelia which weresubsequently frozen in liquid nitrogen. The frozen mycelia were groundto a fine powder in an electric coffee grinder, and the powder was addedto 20 ml of TE buffer and 5 ml of 20% w/v sodium dodecylsulfate (SDS) ina disposable plastic centrifuge tube. The mixture was gently invertedseveral times to ensure mixing, and extracted twice with an equal volumeof phenol:chloroform:isoamyl alcohol (25:24:1 v/v/v). Sodium acetate (3Msolution) was added to give a final concentration of 0.3M and thenucleic acids were precipitated with 2.5 volumes of ice cold ethanol.The tube was centrifuged at 15,000×g for 30 minutes and the pellet wasallowed to air dry for 30 minutes before resuspension in 0.5 ml of TEbuffer. DNase-free ribonuclease A was added to a concentration of 100μg/ml and the mixture was incubated at 37° C. for 30 min. Proteinase K(200 μg/ml) was then added and the mixture was incubated an additionalhour at 37° C. Finally, the mixture was extracted twice withphenol:chloroform:isoamyl alcohol (25:24:1 v/v/v) before precipitatingthe DNA with sodium acetate and ethanol according to standardprocedures. The DNA pellet was dried under vacuum, resuspended in TEbuffer, and stored at 4° C.

Example 7

Hybridization Analysis of Genomic DNA

The total cellular DNA sample prepared as described in Example 6 wasanalyzed by Southern hybridization (Maniatis et al., 1982, MolecularCloning, a Laboratory Manual, Cold Spring Harbor Press, Cold SpringHarbor, N.Y.). Approximately 5 μg of the DNA sample were digested withBamHI, EcoRI, or HindIII and fractionated by size on a 1% agarose gel.The gel was photographed under short wavelength UV light and soaked for15 minutes in 0.5M NaOH-1.5M NaCl followed by 15 minutes in 1M Tris-HCIpH 8-1.5M NaCl. DNA in the gel was transferred onto a Nytran™hybridization membrane (Schleicher & Schuell, Keene, NH) by capillaryblotting in 20 X SSPE (3M sodium chloride-0.2M sodium dibasicphosphate-0.02M disodium EDTA) according to Davis et al. (1980, AdvancedBacterial Genetics, A Manual for Genetic Engineering, Cold Spring HarborPress, Cold Spring Harbor, N.Y.). The membrane was baked for 2 hours at80° C. under vacuum and was soaked for 2 hours in the followinghybridization buffer at 45° C. with gentle agitation: 5 X SSPE, 35%formamide (v/v), 0.3% SDS, and 200 μg/ml denatured and sheared salmontestes DNA. A mutanase-specific probe fragment (approximately 1.8 kb)comprising the coding sequence of a Trichoderma harzianum mutanase cDNA(see, for example, Japanese Patent No. 4-58889/A) was radiolabeled bynick translation (Maniatis et al., supra) with α ³² P!dCTP (Amersham,Arlington Heights, Ill.) and added to the hybridization buffer at anactivity of approximately 1×10⁶ cpm per ml of buffer. The mixture wasincubated with the membrane overnight at 45° C. in a shaking water bath.Following incubation, the membrane was washed once in 0.2 X SSPE with0.1% SDS at 45° C. followed by two washes in 0.2 X SSPE (no SDS) at thesame temperature. The membrane was dried on a paper towel for 15minutes, then wrapped in Saran-Wrap™ and exposed to X-ray film overnightat -70° C. with intensifying screens (Kodak, Rochester, N.Y.).

Southern blotting indicated that the Trichoderma harzianum mutanase cDNAcan be used as a probe under conditions of moderate stringency toidentify and clone the mutanase gene from Penicillium purpurogenum CBS238.95 shown in FIG. 1.

Example 8

DNA Libraries and Identification of Mutanase Clones

Genomic DNA libraries were constructed using the bacteriophage cloningvector λZipLox (Life Technologies, Gaithersburg, Md.) with E. coliY1090ZL cells (Life Technologies, Gaithersburg, Md.) as a host forplating and purification of recombinant bacteriophage and E. coliDH10Bzip (Life Technologies, Gaithersburg, Md.) for excision ofindividual pZL1-mutanase clones. Total cellular DNA was partiallydigested with Tsp509I and size-fractionated on 1% agarose gels. DNAfragments migrating in the size range 3-7 kb were excised and elutedfrom the gel using Prep-a-Gene reagents (BioRad Laboratories, Hercules,Calif). The eluted DNA fragments were ligated with EcoRI-cleaved anddephosphorylated λZipLox vector arms (Life Technologies, Gaithersburg,Md.), and the ligation mixtures were packaged using commercial packagingextracts (Stratagene, La Jolla, Calif.). The packaged DNA libraries wereplated and amplified in Escherichia coli Y1090ZL cells (LifeTechnologies, Gaithersburg, Md.). The unamplified genomic librarycontained 4.1×10⁶ pfu/ml (the control ligation with no genomic DNAinserts yields 2.0×10⁴ pfu/ml). Approximately 45,000 plaques from thelibrary were screened by plaque-hybridization with the radiolabeledTrichoderma harzianum mutanase probe fragment described in Example 7.Eighteen positive clones which hybridize strongly to the probe werepicked and ten were purified twice in E. coli Y1090ZL cells. Themutanase clones were subsequently excised from the λZipLox vector aspZL1-mutanase clones (D'Alessio et al., 1992, Focus® 14:76).

Example 9

DNA Sequence Analysis of Penicillium purpurogenum CBS 238.95 MutanaseGene

Restriction mapping of the pZL1-mutanase clones described in Example 8was performed using standard methods (Maniatis et al., supra). DNAsequencing of the mutanase clones described in Example 8 was performedwith an Applied Biosystems Model 373A Automated DNA Sequencer (AppliedBiosystems, Inc., Foster City, Calif.) using a combination of shotgunDNA sequencing (Messing et al., 1981, Nucleic Acids Research 9:309-321)and the primer walking technique with dye-terminator chemistry (Gieseckeet al., 1992, Journal of Virol. Methods 38:47-60). In addition to thelac-forward and lac-reverse primers, specific oligonucleotide sequencingprimers were synthesized on an Applied Biosystems Model 394 DNA/RNASynthesizer according to the manufacturer's instructions.

Example 10

Properties of the Penicillium purpurogenum CBS 238.95 Mutanase Gene

Restriction mapping of one of the pZL1-mutanase clones designated Pp6A(E. coli INVα1F - pZL-Pp6A) reveals that the region which hybridizesunder conditions of moderate stringency to the Trichoderma harzianummutanase cDNA was localized near one end of a 3.6 kb genomic DNA insertshown in FIG. 2.

DNA sequencing of a portion of this segment shows an open reading frame(SEQ ID NO:2) with homology to the Trichoderma harzianum mutanase cDNAand the deduced amino acid sequence of the Penicillium purpurogenummutanase (SEQ ID NO:3) shown in FIG. 3.

The positions of introns and exons within the Penicillium purpurogenumCBS 238.95 mutanase gene were assigned based on alignments of thededuced amino acid sequence to the corresponding Trichoderma harzianummutanase gene product. On the basis of this comparison, the Penicilliumpurpurogenum CBS 238.95 mutanase gene was comprised of five exons (126,532, 226, 461, and 548 bp) which are interrupted by four small introns(63, 81, 58, and 78 bp). The sizes and composition of the introns areconsistent with those of other fungal genes (Gurr et al., 1987, InKinghorn, J. R. (ed.), Gene Structure in Eukaryotic Microbes, pp.93-139, IRL Press, Oxford) in that all contain consensus splice donorand acceptor sequences as well as the consensus lariat sequence(PuCTPuAC) near the 3' end of each intervening sequence.

A comparison of the N-terminal amino acid sequence described in Example5 with the deduced N-terminal amino acid sequence of the Penicilliumpurpurogenum CBS 238.95 mutanase gene product set forth in FIG. 3 (SEQID NO:3) predicted an amino terminal extension of 30 amino acids whichis not present in the mature enzyme. Based on the rules of von Heijne(von Heijne, 1984, Journal of Molecular Biology 173:243-251), the first20 amino acids likely comprise a secretory signal peptide which directsthe nascent polypeptide into the endoplasmic reticulum. The next 10amino acid residues probably represent a propeptide segment which issubsequently removed by proteolytic cleavage following a dibasic Arg-Argsequence. The mature mutanase is an acidic protein (calculatedisoelectric point=3.8) composed of 600 amino acids (MW=63,443). Sincethe observed molecular weight on SDS-PAGE (ca. 96,000) is considerablygreater than that predicted by the deduced amino acid sequence set forthin FIG. 3 (SEQ ID NO:3), it appears likely that the mutanase contains aconsiderable amount of carbohydrate, possibly as much as 34% by weight.The signal peptide and propeptide portions of the Penicilliumpurpurogenum mutanase share little similarity with the Trichodermaharzianum mutanase shown in FIG. 4 (SEQ ID NO:5).

The deduced amino acid sequence of the mature Penicillium purpurogenumCBS 238.95 mutanase shares approximately 52.8% identity with themutanase from Trichoderma harzianum (Japanese Patent No. 4-58889/A)shown in FIG. 4 (SEQ ID NO:5). The regions of greatest identity arelocated in the amino terminal half of these two proteins as well as overthe last 70 residues comprising their respective C-termini. The maturePenicillium purpurogenum mutanase appears to be comprised of threedistinct domains: (1) an amino terminal catalytic domain, (2) a Ser-Thrrich linker domain, and (3) a C-terminal polysaccharide (i.e., mutan)binding domain (residues 548-630). The Ser-Thr rich domain (residues475-547) is composed of 62% Ser and Thr, and is bordered roughly by Cysresidues at positions 477 and 547. This region may be heavilyglycosylated (O-linked) in a manner similar to the Ser-Thr rich linkerregion of Aspergillus niger glucoamylase (Coutinho and Reilly, 1994,Protein Engineering 7:393-400).

Example 11

Expression of Penicillium purpurogenum CBS 238.95 mutanase inAspergillus oryzae

Two synthetic oligonucleotide primers shown below were designed toamplify the Penicillium purpurogenum CBS 238.05 mutanase gene fromplasmid pZL-Pp6A.

5'-cccatttaaatATGAAAGTCTCCAGTGCCTTC-3' (SEQ ID NO:6)

5'-cccttaattaaTTAGCTCTCTACTTGACAAGC-3' (SEQ ID NO:7)

20 (capital letters correspond to the sequence present in the mutanasegene)

One hundred picomoles of each of the primers were used in a PCR reactioncontaining 52 ng plasmid DNA, 1×Pwo Buffer (Boehringer Mannheim,Indianapolis, Ind.), 1 mM each dATP, dTTP, dGTP, and dCTP, and 2.5 unitsof PwoI (Boehringer Mannheim, Indianapolis, Ind.). The amplificationconditions were one cycle at 95° C. for 3 minutes, 25 cycles each at 95°C. for 1 minute, 60° C. for 1 minute, and 72° C. for 1.5 minutes, and afinal cycle at 72° C. for 5 minutes. The amplified 2.2 kb DNA fragmentwas purified by gel electrophoresis and cut with restrictionendonucleases SwaI and PacI (using conditions specified by themanufacturer). The cut fragment was cloned into plasmid pBANe6 (FIG. 5)that had been previously cut with SwaI and PacI resulting in theexpression plasmid pJeRS35.

Plasmid pJeRS35 was introduced into an alkaline protease-deficientAspergillus oryzae host JaL142-6 using standard protoplasttransformation methods (Christensen et al. 1988. Bio/Technology1419-1422). The transformation was conducted with protoplasts at aconcentration of ca. 2×10⁷ protoplasts per ml. One hundred μl ofprotoplasts were placed on ice with ca. 5 μg DNA for 30 minutes. One mlof SPTC (40% PEG 4000, 0.8M sorbitol, 0.05M Tris pH 8.0, 0.05M CaCl₂)was added and the protoplasts were incubated at room temperature for 20minutes. Seven ml Cove agar overlay (per liter: 0.52 g of KCI, 0.52 g ofMgSO₄ -7H₂ 0, 1.52 g of KH₂ PO₄, 1 ml of trace metals described below,0.8M sucrose, and 1% low melt agar) were added to the transformationprior to plating onto COVE transformation plates (per liter: 0.52 g ofKCI, 0.52 g of MgSO₄ -7H₂ 0, 1.52 g of KH₂ PO₄, 1 ml of trace metalsdescribed below, 342.3 g of sucrose, 25 g of Noble agar, 10 ml of 1Macetamide, 10 ml of 3M CsCl). The trace metals solution (1000×) iscomprised of 22 g of ZnSO₄ -7H₂ O, 11 g of H₃ BO₃, 5 g of MnCl₂ -4H₂ O,5 g of FeSO₄ -7H₂ O, 1.6 g of CoCl₂ -5H₂ O, 1.6 g of (NH₄)₆ Mo₇ O₂₄, and50 g of Na₄ EDTA per liter. Plates were incubated 5-7 days at 34° C.Transformants were transferred to plates of the same medium andincubated 3-5 days at 37° C. The transformants were purified bystreaking spores and picking isolated colonies using the same platesunder the same conditions. Totally, 40 transformants were recovered isby their ability to grow on COVE medium using acetamide as sole nitrogensource.

The transformants were grown for 3 days at 34° C. with agitation inshake flasks containing 20 ml of MY50N medium comprised of 62 g ofNutriose, 2.0 g of MgSO₄ -7H₂ O, 2.0 g of KH₂ PO₄, 4.0 g of citric acid,8.0 g of yeast extract, 2.0 g of urea, 0.1 g of CaC1₂, and 0.5 ml oftrace metals solution per liter adjusted to pH 6.0. The trace metalssolution consisted of 2.2 g of ZnSO₄ x7H₂ O, 1.1 g of H₃ BO₃, 0.5 g ofMnCl₂ -4H₂ O, 0.5 g of FeSO₄ x7H₂ O, 0.16 g of CoCl₂ x5H₂ O, 0.16 g of(NH₄)₆ Mo₇ O₂₄, and 5 g of Na₄ EDTA per 100 ml of deionized water.

Mutan assay plates were prepared by blending a suspension of 1% (v/w)mutan, 1% agarose in 0.1M sodium acetate pH 5.5 buffer for 20 minutes at4° C. The agarose was melted by heating and 150 mm petri plates werepoured. After solidification, small wells (ca. 40 μl equivalent volume)were punched in the plates. Thirty-five μl volumes of centrifuged brothof the 40 grown transformant cultures (and one untransformed control)were pipetted into the wells and the plates were incubated at 37° C.After overnight incubation, 14 of the transformant wells showed opaqueclearing zones (the control showed no clearing zone).

The broths from the positive transformants were analyzed by SDS-PAGEusing 8-16% polyacrylamide Novex gels (Novex, San Diego, Calif.)according to the manufacturer's instructions. The transformants showed aprominent band at ca. 96 kDa while no band of this size was observedfrom the broth of the control culture. The 96 kDa band from one of thetransformant cultures was re-isolated by SDS-PAGE and blot transferredto PVDF membrane (Novex, San Diego, Calif.) using 10 mM CAPS (3-cyclohexylamino!-1-propanesulfonic acid) in 10% Methanol, pH=11.0 for 2hours. The PVDF membrane was stained with 0.1% Coomassie® Blue R-250 in40% MeOH/1% acetic acid for 20 seconds. The stained band was excised andsubjected to N-terminal sequencing on a Applied Biosystems Inc. Model476A protein sequencer (Applied Biosystems, Foster City, Calif.) using ablot cartridge and liquid phase trifluoroacetic acid delivery accordingto manufacturer's instructions. The results showed the expectedN-terminus of the mutanase based on the DNA sequence. N-terminalprocessing followed a Kex-2 cleavage site. The N-terminal sequence wasdetermined to be STSDRLVFAHFMVGIVSDRTSA (SEQ ID NO:1).

Example 12

Purification and characterization of recombinant Penicilliumpurpurogenum mutanase

One of the transformants described in Example 11, Aspergillus oryzaeJeRS323, was grown at 30° C., 200 rpm for 4 days in 1.0 liter shakeflasks containing 250 ml of a medium consisting of of 10 g of yeastextract and 20 g of peptone per liter supplemented with 2% maltose. Thewhole culture broths were filtered through Miracloth.

Mutan, prepared as described in Example 2, was washed with 0.1M sodiumacetate pH 5.5 buffer and then suspended in an amount of 15.6 g to 780ml of 0.45 μm filtered shake flask broth to provide a 2% solution. Thesuspension was adjusted to pH 5.5 and then mixed at 4° C. for 1 hour.The suspension was then filtered on a sintered glass filter funnel,washed 4 times with 0.1M sodium acetate pH 5.5 buffer (total volume:1110 ml), and finally 6 times with deionized water (total volume: 1250ml). After each washing step, the suspension was filtered and thefiltrate fractions collected. Elution of the mutanase was determined bymeasuring production of soluble reducing sugars released from mutan.Specifically, 0.1 ml of 5% mutan in 50 mM sodium acetate pH 5.5 buffer(allowed to swell at least for 1 hour) was added to 0.3 ml of eachfraction (diluted in deionized water) in round bottomed Eppendorf vials(to ensure sufficient agitation) and incubated for 15 minutes at 40° C.with vigorous shaking. The reaction was terminated by adding 0.1 ml of0.4M NaOH. The samples were centrifuged, filtered through 0.45 μmfilters (Millipore, Bedford, Mass.), and the filtrates collected. Avolume of 100 μl of each filtrate were added to 750 μl of ferricyanidereagent (0.4 g/l K₃ Fe(CN)₆, 20 g/l Na₂ CO₃) in Eppendorf vials andincubated 15 minutes at 85° C. After allowing the samples to cool, thedecrease in absorption at 420 nm was measured. A dilution series ofglucose was included as a standard. Substrate and enzyme blanks wereincluded as controls. Samples were run in duplicates. One mutanase unit(MU) is defined as the amount of enzyme which produces 1 μmole ofreducing sugars (measured as glucose equivalents) per minute from mutanat pH 5.5 and 40° C.

The recombinant mutanase eluted during the washing with water. Thefiltrates were pooled, 0.7 μm filtered (Whatman, Fairfield, N.J.),concentrated on a Microsep™ Microconcentrator (Filtron, Northborough,Mass.) equipped with a 10 kDa MW-CO membrane, and further concentratedto 25 ml on an Amicon cell equipped with a YM10 membrane (Amicon,Beverly, Mass.). The purification resulted in a 129 fold purificationwith a yield of around 20% (Table 1). The relative low yield can beexplained by an incomplete adsorption on the mutan and some leakage ofmutanase during the washing steps. The purity of the mutanase wasestimated to be >95% by SDS-PAGE and IEF with a molecular weight around90 kDa and an isolectric point (pI) of approximately pH 3 (theoreticalpI=3.95). The N-terminal amino acid sequence was verified to beSer-Thr-Ser-Asp-Arg- (SEQ ID NO: 1).

                  TABLE 1                                                         ______________________________________                                        Purification of recombinant Penicillium purpurogenum mutanase                                                     Total                                                                  Activity                                                                             Activity                                  Sample                                                                              Volume (ml)                                                                             A.sub.280                                                                             A.sub.260                                                                          (MU/ml)                                                                              (MU)  Yield (%)                           ______________________________________                                        Broth 780       19.8    27.3 2.2    1716  100                                 Purified                                                                            25        0.90    0.65 12.8   320   19                                  ______________________________________                                    

Temperature profiles were obtained by incubating the assay mixture (50mM sodium acetate pH 5.5 or 50 mM sodium phosphate pH 7 buffer) usingthe procedure above at various temperatures. pH profiles were obtainedby suspending the mutan in 50 mM buffer at various pH (glycine-HC1 forpH 3-3.5, sodium acetate for pH 4-5.5, and sodium phosphate for pH6-7.5).

The pH- and temperature-profiles for the purified recombinantPenicillium purpurogenum mutanase are shown in FIGS. 6 and 7,respectively. The enzyme exhibits a fairly broad pH optimum around pH3.5-5 and temperature optimum around 40°-45° C. at pH 7 and 50°-55° C atpH 5.5.

Binding isotherms were obtained by incubating various concentrations ofthe purified recombinant Penicillium purpurogenum mutanase in a 0. 2%suspension of mutan in 0.1M sodium phosphate pH 7 buffer for 1 hour at4° C. with stirring. Samples were then centrifuged for 10 minutes at15000×g and the amount of enzyme left in the supernatant determined byfluorescence spectrometry using a Perkin Elmer LS50 fluorescencespectrometer with excitation at 280 nm and emission at 345 nm. Afluorescence standard curve was constructed based on the purifiedmutanase.

The binding isotherm observed for the purified recombinant Penicilliumpurpurogenum mutanase binding to mutan could be fitted using the simpleLangmuir model for adsorption on solid surfaces. The Penicilliumpurpurogenum mutanase show similar strong affinity for the mutan with adesorption constant (K_(d)) around 0.111±0.016 μM and a maximum bindingcapacity (A_(max)) of 0.244±0.012 μmol enzyme/g mutan.

Differential scanning calorimetry of the purified recombinantPenicillium purpurogenum mutanase was performed using a MicroCal MC-2instrument according to the manufacturer's instructions. The scan wasperformed from 5° C. to 95° C. at a constant scan rate of 90 C per hour.A midpoint denaturation temperature of around 46° C. at pH 7 wasobserved for the Penicillium purpurogenum mutanase.

Deposit of Biological Materials

The following strain has been deposited according to the Budapest Treatyin the Agricultural Research Service Patent Culture Collection (NRRL),Northern Regional Research Laboratory, 1815 University Street, Peoria,Ill. 61604, USA.

    ______________________________________                                        Strain         Accession Number                                                                           Deposit Date                                      ______________________________________                                        E. coli INVα1F (pZL-Pp6A)                                                              NRRL B-21518 January 18, 1996                                  ______________________________________                                    

The strain has been deposited under conditions that assure that accessto the culture will be available during the pendency of this patentapplication to one determined by the Commissioner of Patents andTrademarks to be entitled thereto under 37 C. F. R. ¤1.14 and 35 U. S.C. ¤122. The deposit represents a substantially pure culture of eachdeposited strain. The deposit is available as required by foreign patentlaws in countries wherein counterparts of the subject application, orits progeny are filed. However, it should be understood that theavailability of a deposit does not constitute a license to practice thesubject invention in derogation of patent rights granted by governmentalaction.

The invention described and claimed herein is not to be limited in scopeby the specific embodiments herein disclosed, since these embodimentsare intended as illustrations of several aspects of the invention. Anyequivalent embodiments are intended to be within the scope of thisinvention. Indeed, various modifications of the invention in addition tothose shown and described herein will become apparent to those skilledin the art from the foregoing description. Such modifications are alsointended to fall within the scope of the appended claims.

Various references are cited herein, the disclosures of which areincorporated by reference in their entireties.

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 7                                                  (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       SerThrSerAspArgLeuValPheAlaHisPheMetValGlyIleVal                              151015                                                                        SerAspArgThrSerAla                                                            20                                                                            (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 2523 base pairs                                                   (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: join(41..166, 230..760, 842..1069, 1128..1586,                  1665..2210)                                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: sig.sub.-- peptide                                              (B) LOCATION: 41..130                                                         (ix) FEATURE:                                                                 (A) NAME/KEY: mat.sub.-- peptide                                              (B) LOCATION: join(131..166, 230..760, 842..1069, 1128..1586,                 1665..2210)                                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       AATTGTGCCCTAAACCTCCTCCTGGAGGAACACACTCAAGATGAAAGTCTCCAGT55                     MetLysValSerSer                                                               30                                                                            GCCTTCGCGGCGACGCTGTCCGCAATTATAGCTGCGTGCTCAGCTCTT103                           AlaPheAlaAlaThrLeuSerAlaIleIleAlaAlaCysSerAlaLeu                              25-20-15-10                                                                   CCTTCTGACTCAATGGTTTCGAGGCGAAGCACATCGGACCGTCTCGTG151                           ProSerAspSerMetValSerArgArgSerThrSerAspArgLeuVal                              515                                                                           TTCGCGCATTTCATGGTAAACATCCATCTCGAATATGAGGCACATAGTCAGTGAC206                    PheAlaHisPheMet                                                               10                                                                            GATAGATTGGCTGACTTCATCAGGTTGGTATCGTCAGTGACCGGACCAGT256                         ValGlyIleValSerAspArgThrSer                                                   1520                                                                          GCTAGCGATTATGACGCCGACATGCAGGGTGCTAAAGCTTATGGAATT304                           AlaSerAspTyrAspAlaAspMetGlnGlyAlaLysAlaTyrGlyIle                              253035                                                                        GACGCCTTTGCATTGAATATCGGTACCGATACCTTCAGCGACCAGCAA352                           AspAlaPheAlaLeuAsnIleGlyThrAspThrPheSerAspGlnGln                              404550                                                                        CTGGGGTATGCCTACGAGTCTGCGGCAAACAATGACATGAAAGTGTTC400                           LeuGlyTyrAlaTyrGluSerAlaAlaAsnAsnAspMetLysValPhe                              556065                                                                        ATTTCATTCGATTTCAACTGGTGGTCCACCAGCCAGGCCACCGAAATT448                           IleSerPheAspPheAsnTrpTrpSerThrSerGlnAlaThrGluIle                              70758085                                                                      GGCCAAAAGATTGCCCAGTACGGTAGCCTACCAGGCCAGCTCATGTAT496                           GlyGlnLysIleAlaGlnTyrGlySerLeuProGlyGlnLeuMetTyr                              9095100                                                                       GATGACAAGATTTTCGTCTCGTCGTTTGCTGGCGACGGTGTAGACGTG544                           AspAspLysIlePheValSerSerPheAlaGlyAspGlyValAspVal                              105110115                                                                     GCAGCATTGAAGTCAGCTGCTGGCGGCAATGTGTTCTTCGCTCCAAAC592                           AlaAlaLeuLysSerAlaAlaGlyGlyAsnValPhePheAlaProAsn                              120125130                                                                     TTCCATCCATCGTATGGTACAGACCTGTCGGATGTCGATGGTCTTCTC640                           PheHisProSerTyrGlyThrAspLeuSerAspValAspGlyLeuLeu                              135140145                                                                     AACTGGATGGGCTGGCCTAGCAATGGTAATAACAAGGCTCCAACTGCC688                           AsnTrpMetGlyTrpProSerAsnGlyAsnAsnLysAlaProThrAla                              150155160165                                                                  GGTGCCAACGTTACCGTTGAGGAAGGGGACGAGGAATATATAACTGCT736                           GlyAlaAsnValThrValGluGluGlyAspGluGluTyrIleThrAla                              170175180                                                                     TTGGATGGCAAGCCCTACATTGCTGTCAGTCGCCTAACCCTACCTCCTAGCCTT790                     LeuAspGlyLysProTyrIleAla                                                      185                                                                           GGAGCAAAACGATTCAGTTTGGCTGACCTTTTCTTTTTTCTTCTTCACTAGCCGGCC847                  ProAla                                                                        190                                                                           TCACCATGGTTCTCTACGCATTTTGGGCCAGAGGTGACATACAGCAAG895                           SerProTrpPheSerThrHisPheGlyProGluValThrTyrSerLys                              195200205                                                                     AACTGGGTTTTCCCATCTGATTTGCTTTTCTACCAGCGTTGGAATGAT943                           AsnTrpValPheProSerAspLeuLeuPheTyrGlnArgTrpAsnAsp                              210215220                                                                     CTATTGAATTTGGGCCCTCAATTCATTGAAGTGGTCACCTGGAATGAC991                           LeuLeuAsnLeuGlyProGlnPheIleGluValValThrTrpAsnAsp                              225230235                                                                     TATGGTGAATCGCAATATGTCGGACCTCTGAACTCTCCTCATACAGAC1039                          TyrGlyGluSerGlnTyrValGlyProLeuAsnSerProHisThrAsp                              240245250255                                                                  GATGGCTCCTCTCGATGGGCGAATGACATGGTAAGCCATCTTGTGTAGGT1089                        AspGlySerSerArgTrpAlaAsnAspMet                                                260265                                                                        ATCGGTGTTTTGTTTCTATGCTAACATCAAGAAACTAGCCTCACGATGGCTGG1142                     ProHisAspGlyTrp                                                               270                                                                           CTGGATCTGGCAAAGCCCTACATCGCGGCATTCCACGACGGGGCCACT1190                          LeuAspLeuAlaLysProTyrIleAlaAlaPheHisAspGlyAlaThr                              275280285                                                                     TCGCTATCATCATCCTACATCACCGAAGACCAGCTCATCTACTGGTAT1238                          SerLeuSerSerSerTyrIleThrGluAspGlnLeuIleTyrTrpTyr                              290295300                                                                     CGGCCTCAACCACGACTCATGGACTGCGACGCAACTGATACCTGCATG1286                          ArgProGlnProArgLeuMetAspCysAspAlaThrAspThrCysMet                              305310315                                                                     GTTGCTGCCAACAATGACACGGGCAACTATTTCGAGGGCAGACCCAAT1334                          ValAlaAlaAsnAsnAspThrGlyAsnTyrPheGluGlyArgProAsn                              320325330                                                                     GGGTGGGAAAGCATGGAGGACGCTGTCTTCGTGGTTGCTTTGCTCCAG1382                          GlyTrpGluSerMetGluAspAlaValPheValValAlaLeuLeuGln                              335340345350                                                                  TCTGCTGGAACGGTTCAGGTCACTTCAGGCCCTAATACCGAGACATTT1430                          SerAlaGlyThrValGlnValThrSerGlyProAsnThrGluThrPhe                              355360365                                                                     GATGCTCCTGCTGGTGCAAGCGCCTTCCAGGTTCCCATGGGCTTCGGC1478                          AspAlaProAlaGlyAlaSerAlaPheGlnValProMetGlyPheGly                              370375380                                                                     CCCCAGAGCTTCTCCCTGTCGCGGGATGGCGAGACAGTATTGTCTGGA1526                          ProGlnSerPheSerLeuSerArgAspGlyGluThrValLeuSerGly                              385390395                                                                     ACAAGCTTGAAGGATATCATTGATGGATGCTTGTGCGGAATCTACAAC1574                          ThrSerLeuLysAspIleIleAspGlyCysLeuCysGlyIleTyrAsn                              400405410                                                                     TTCAACGCCTATGTAAGAACTGCCGTGTCTTTTGTATATCTGAATATGTTTC1626                      PheAsnAlaTyr                                                                  415                                                                           CAAGGTTATTGACATGGGAAAAAAAAAAAAAAATTCAGGTGGGCTCTCTGCCA1679                     ValGlySerLeuPro                                                               420                                                                           GCAACTTTCTCCGATCCACTCGAGCCACCTTCTCTCAACGCCTTCAGC1727                          AlaThrPheSerAspProLeuGluProProSerLeuAsnAlaPheSer                              425430435                                                                     GAAGGCTTGAAGGTTTCGACATGCAGCGCGACACCATCTTTGGGATTG1775                          GluGlyLeuLysValSerThrCysSerAlaThrProSerLeuGlyLeu                              440445450455                                                                  ACATCGACCACTCCACCAGAGACCATTCCTACAGGCACGATTACTCCA1823                          ThrSerThrThrProProGluThrIleProThrGlyThrIleThrPro                              460465470                                                                     GGATCAGCTATTACAGGTGCTGCAACAACTACCTCTACCATCTCGACC1871                          GlySerAlaIleThrGlyAlaAlaThrThrThrSerThrIleSerThr                              475480485                                                                     ACCTCCACGATTTCCACGACCTCAACTTTTATCTCAACTACCACCACC1919                          ThrSerThrIleSerThrThrSerThrPheIleSerThrThrThrThr                              490495500                                                                     ACCACGTCCAGTGCTGCTACCTCCACCACCACCGGAACTTGCATCGCC1967                          ThrThrSerSerAlaAlaThrSerThrThrThrGlyThrCysIleAla                              505510515                                                                     GGCACTGGCCCTGACAACTATTCTGGCCTGTGTTCCTTCTGCTGTAAC2015                          GlyThrGlyProAspAsnTyrSerGlyLeuCysSerPheCysCysAsn                              520525530535                                                                  TACGGCTACTGTCCGGGCTCCGATGGTTCGGCCGGCCCGTGTACATGC2063                          TyrGlyTyrCysProGlySerAspGlySerAlaGlyProCysThrCys                              540545550                                                                     ACGGCCTATGGAGATCCAGTTCCTACGCCTCCAGTAACAGGAACAGTT2111                          ThrAlaTyrGlyAspProValProThrProProValThrGlyThrVal                              555560565                                                                     GGCGTTCCGCTTGATGGCGAGGGTGACAGTTACTTGGGTCTGTGTAGT2159                          GlyValProLeuAspGlyGluGlyAspSerTyrLeuGlyLeuCysSer                              570575580                                                                     TTTGCCTGCAACCACGGCTATTGCCCGTCTACTGCTTGTCAAGTAGAG2207                          PheAlaCysAsnHisGlyTyrCysProSerThrAlaCysGlnValGlu                              585590595                                                                     AGCTGAGAGGTGCCACTATCTAGGTAATACCATGTTAAAGTAATACCTAGGTA2260                     Ser                                                                           600                                                                           CTCTGTGTCTAGCTTGAGAGATGGCAGGGTATCTAGTTCTATCTTAAATATAAGATTTCT2320              CCAACTTACATGATTTTGATGCACATGGATAGGTAGACCTGGACAGTGAAGGGCAATACT2380              TAAATAATGCAAACAGACACTGGATCTATATCGTTCAACTCAGTTGGCCAAAGACTAGTC2440              GTGAAAAAAACACCCTTTCGAACAAAAACCTTCTTCGCTGCATCAACGCAGTCCAAAATA2500              AGTCCAATCCCCTCCACCATGAA2523                                                   (2) INFORMATION FOR SEQ ID NO:3:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 630 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                       MetLysValSerSerAlaPheAlaAlaThrLeuSerAlaIleIleAla                              30-25-20-15                                                                   AlaCysSerAlaLeuProSerAspSerMetValSerArgArgSerThr                              10-51                                                                         SerAspArgLeuValPheAlaHisPheMetValGlyIleValSerAsp                              51015                                                                         ArgThrSerAlaSerAspTyrAspAlaAspMetGlnGlyAlaLysAla                              202530                                                                        TyrGlyIleAspAlaPheAlaLeuAsnIleGlyThrAspThrPheSer                              35404550                                                                      AspGlnGlnLeuGlyTyrAlaTyrGluSerAlaAlaAsnAsnAspMet                              556065                                                                        LysValPheIleSerPheAspPheAsnTrpTrpSerThrSerGlnAla                              707580                                                                        ThrGluIleGlyGlnLysIleAlaGlnTyrGlySerLeuProGlyGln                              859095                                                                        LeuMetTyrAspAspLysIlePheValSerSerPheAlaGlyAspGly                              100105110                                                                     ValAspValAlaAlaLeuLysSerAlaAlaGlyGlyAsnValPhePhe                              115120125130                                                                  AlaProAsnPheHisProSerTyrGlyThrAspLeuSerAspValAsp                              135140145                                                                     GlyLeuLeuAsnTrpMetGlyTrpProSerAsnGlyAsnAsnLysAla                              150155160                                                                     ProThrAlaGlyAlaAsnValThrValGluGluGlyAspGluGluTyr                              165170175                                                                     IleThrAlaLeuAspGlyLysProTyrIleAlaProAlaSerProTrp                              180185190                                                                     PheSerThrHisPheGlyProGluValThrTyrSerLysAsnTrpVal                              195200205210                                                                  PheProSerAspLeuLeuPheTyrGlnArgTrpAsnAspLeuLeuAsn                              215220225                                                                     LeuGlyProGlnPheIleGluValValThrTrpAsnAspTyrGlyGlu                              230235240                                                                     SerGlnTyrValGlyProLeuAsnSerProHisThrAspAspGlySer                              245250255                                                                     SerArgTrpAlaAsnAspMetProHisAspGlyTrpLeuAspLeuAla                              260265270                                                                     LysProTyrIleAlaAlaPheHisAspGlyAlaThrSerLeuSerSer                              275280285290                                                                  SerTyrIleThrGluAspGlnLeuIleTyrTrpTyrArgProGlnPro                              295300305                                                                     ArgLeuMetAspCysAspAlaThrAspThrCysMetValAlaAlaAsn                              310315320                                                                     AsnAspThrGlyAsnTyrPheGluGlyArgProAsnGlyTrpGluSer                              325330335                                                                     MetGluAspAlaValPheValValAlaLeuLeuGlnSerAlaGlyThr                              340345350                                                                     ValGlnValThrSerGlyProAsnThrGluThrPheAspAlaProAla                              355360365370                                                                  GlyAlaSerAlaPheGlnValProMetGlyPheGlyProGlnSerPhe                              375380385                                                                     SerLeuSerArgAspGlyGluThrValLeuSerGlyThrSerLeuLys                              390395400                                                                     AspIleIleAspGlyCysLeuCysGlyIleTyrAsnPheAsnAlaTyr                              405410415                                                                     ValGlySerLeuProAlaThrPheSerAspProLeuGluProProSer                              420425430                                                                     LeuAsnAlaPheSerGluGlyLeuLysValSerThrCysSerAlaThr                              435440445450                                                                  ProSerLeuGlyLeuThrSerThrThrProProGluThrIleProThr                              455460465                                                                     GlyThrIleThrProGlySerAlaIleThrGlyAlaAlaThrThrThr                              470475480                                                                     SerThrIleSerThrThrSerThrIleSerThrThrSerThrPheIle                              485490495                                                                     SerThrThrThrThrThrThrSerSerAlaAlaThrSerThrThrThr                              500505510                                                                     GlyThrCysIleAlaGlyThrGlyProAspAsnTyrSerGlyLeuCys                              515520525530                                                                  SerPheCysCysAsnTyrGlyTyrCysProGlySerAspGlySerAla                              535540545                                                                     GlyProCysThrCysThrAlaTyrGlyAspProValProThrProPro                              550555560                                                                     ValThrGlyThrValGlyValProLeuAspGlyGluGlyAspSerTyr                              565570575                                                                     LeuGlyLeuCysSerPheAlaCysAsnHisGlyTyrCysProSerThr                              580585590                                                                     AlaCysGlnValGluSer                                                            595600                                                                        (2) INFORMATION FOR SEQ ID NO:4:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 16 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                       SerSerAlaAspArgLeuValPheCysHisPheMetIleGlyIleVal                              151015                                                                        (2) INFORMATION FOR SEQ ID NO:5:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 635 amino acids                                                   (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                       MetLeuGlyValPheArgArgLeuArgLeuGlyAlaLeuAlaAlaAla                              151015                                                                        AlaLeuSerSerLeuGlySerAlaAlaProAlaAsnValAlaIleArg                              202530                                                                        SerLeuGluGluArgAlaSerSerAlaAspArgLeuValPheCysHis                              354045                                                                        PheMetIleGlyIleValGlyAspArgGlySerSerAlaAspTyrAsp                              505560                                                                        AspAspMetGlnArgAlaLysAlaAlaGlyIleAspAlaPheAlaLeu                              65707580                                                                      AsnIleGlyValAspGlyTyrThrAspGlnGlnLeuGlyTyrAlaTyr                              859095                                                                        AspSerAlaAspArgAsnGlyMetLysValPheIleSerPheAspPhe                              100105110                                                                     AsnTrpTrpSerProGlyAsnAlaValGlyValGlyGlnLysIleAla                              115120125                                                                     GlnTyrAlaAsnArgProAlaGlnLeuTyrValAspAsnArgProPhe                              130135140                                                                     AlaSerSerPheAlaGlyAspGlyLeuAspValAsnAlaLeuArgSer                              145150155160                                                                  AlaAlaGlySerAsnValTyrPheValProAsnPheHisProGlyGln                              165170175                                                                     SerSerProSerAsnIleAspGlyAlaLeuAsnTrpMetAlaTrpAsp                              180185190                                                                     AsnAspGlyAsnAsnLysAlaProLysProGlyGlnThrValThrVal                              195200205                                                                     AlaAspGlyAspAsnAlaTyrLysAsnTrpLeuGlyGlyLysProTyr                              210215220                                                                     LeuAlaProValSerProTrpPhePheThrHisPheGlyProGluVal                              225230235240                                                                  SerTyrSerLysAsnTrpValPheProGlyGlyProLeuIleTyrAsn                              245250255                                                                     ArgTrpGlnGlnValLeuGlnGlnGlyPheProMetValGluIleVal                              260265270                                                                     ThrTrpAsnAspTyrGlyGluSerHisTyrValGlyProLeuLysSer                              275280285                                                                     LeuHisPheAspAspGlyAsnSerLysTrpValAsnAspMetProHis                              290295300                                                                     AspGlyPheLeuAspLeuSerLysProPheIleAlaAlaTyrLysAsn                              305310315320                                                                  ArgAspThrAspIleSerLysTyrValGlnAsnGluGlnLeuValTyr                              325330335                                                                     TrpTyrArgArgAsnLeuLysAlaLeuAspCysAspAlaThrAspThr                              340345350                                                                     ThrSerAsnArgProAlaAsnAsnGlySerGlyAsnTyrPheGluGly                              355360365                                                                     ArgProAspGlyTrpGlnThrMetAspAspAlaValTyrValAlaAla                              370375380                                                                     LeuLeuLysThrAlaGlySerValThrIleThrSerGlyGlyThrThr                              385390395400                                                                  GlnThrPheGlnAlaAsnAlaGlyAlaAsnLeuPheGlnIleProAla                              405410415                                                                     SerIleGlyGlnGlnLysPheAlaLeuThrArgAsnGlyGlnThrIle                              420425430                                                                     PheSerGlyThrSerLeuMetAspIleThrAsnValCysSerCysGly                              435440445                                                                     IleTyrAsnPheAsnProTyrValGlyThrIleProAlaGlyPheAsp                              450455460                                                                     AspProLeuGlnAlaAspGlyLeuPheSerLeuThrIleGlyLeuHis                              465470475480                                                                  ValThrThrCysGlnAlaLysProSerLeuGlyThrAsnProProVal                              485490495                                                                     ThrSerGlyProValSerSerLeuProAlaSerSerThrThrArgAla                              500505510                                                                     SerSerProProProValSerSerThrArgValSerSerProProVal                              515520525                                                                     SerSerProProValSerArgThrSerSerProProProProProAla                              530535540                                                                     SerSerThrProProSerGlyGlnValCysValAlaGlyThrValAla                              545550555560                                                                  AspGlyGluSerGlyAsnTyrIleGlyLeuCysGlnPheSerCysAsn                              565570575                                                                     TyrGlyTyrCysProProGlyProCysLysCysThrAlaPheGlyAla                              580585590                                                                     ProIleSerProProAlaSerAsnGlyArgAsnGlyCysProLeuPro                              595600605                                                                     GlyGluGlyAspGlyTyrLeuGlyLeuCysSerPheSerCysAsnHis                              610615620                                                                     AsnTyrCysProProThrAlaCysGlnTyrCys                                             625630635                                                                     (2) INFORMATION FOR SEQ ID NO:6:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 32 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                       CCCATTTAAATATGAAAGTCTCCAGTGCCTTC32                                            (2) INFORMATION FOR SEQ ID NO:7:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 32 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                                       CCCTTAATTAATTAGCTCTCTACTTGACAAGC32                                            __________________________________________________________________________

What is claimed is:
 1. An isolated polypeptide having mutanase activityobtained from a Penicillium purpurogenum strain which(a) has a pHoptimum of about 3.0 to about 4.5 at 40° C.; (b) has a temperatureoptimum throughout the range of 45° C. to 55° C. at pH 5.5; and (c) isencoded by a nucleic acid sequence which hybridizes under low stringencyconditions with (i) the nucleic acid sequence of SEQ ID NO:2, or (ii)its complementary strand.
 2. The polypeptide of claim 1, which has theamino acid sequence of SEQ ID NO:3, or a fragment thereof which hasmutanase activity.
 3. The polypeptide of claim 2, which has the aminoacid sequence of SEQ ID NO:3.
 4. The polypeptide of claim 2, which hasthe amino acid sequence of residues 31-630 of SEQ ID NO:3.
 5. Thepolypeptide of claim 1, which is encoded by a nucleic acid sequencewhich hybridizes under medium stringency conditions with (i) the nucleicacid sequence of SEQ ID NO:2, or (ii) its complementary strand.
 6. Thepolypeptide of claim 5, which is encoded by a nucleic acid sequencewhich hybridizes under high stringency conditions with (i) the nucleicacid sequence of SEQ ID NO:2, or (ii) its complementary strand.
 7. Thepolypeptide of claim 1, which is encoded by the nucleic acid sequencecontained in plasmid pZL-Pp6A which is contained in Escherichia coliNRRL B-21518.
 8. An oral cavity composition comprising the polypeptideof claim 1 and an orally acceptable carrier.